c Indian Academy of Sciences
RESEARCH ARTICLE
Evaluation of point mutations in dystrophin gene in Iranian Duchenne
and Becker muscular dystrophy patients: introducing three
novel variants
MARYAM HAGHSHENAS1 , MOHAMMAD TAGHI AKBARI2,3 ∗ , SHOHREH ZARE KARIZI3,4 ,
FARAVAREH KHORDADPOOR DEILAMANI3,5 , SHAHRIAR NAFISSI6 and ZIVAR SALEHI1
1
2
Faculty of Sciences, Department of Biology, University of Guilan, Rasht 4199613776, Iran
Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-111, Iran
3
Tehran Medical Genetics Laboratory, No. 251, Taleghani Street, Tehran 1598618133, Iran
4
Department of Biology, Varamin Pishva Branch, Azad University, Varamin Pishva 7489-33817, Iran
5
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran 1477892855, Iran
6
Department of Neurology, Tehran University of Medical Sciences (TUMS), Tehran 1417614418, Iran
Abstract
Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions
or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any
large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent
probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50–79. Also exon 44 was
sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four
nonsense, one frameshift and two splice site mutations as well as two missense variants.
[Haghshenas M., Akbari M. T., Zare Karizi S., Khordadpoor Deilamani F., Nafissi S. and Salehi Z. 2016 Evaluation of point mutations in
dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants. J. Genet. 95, 325–329]
Introduction
Duchenne and Becker muscular dystrophies (DMD and
BMD) are X-linked recessive neuromuscular disorders
occurring almost 1 in 3500 and 30000 newborn males,
respectively (Hegde et al. 2008; Flanigan et al. 2011) and
are both distinguished by progressive symmetrical muscular weakness. Clinical symptoms of DMD usually develop
before the age of five resulting in wheelchair dependency
by age 12. Patients usually succumb to the disorder in early
twenties, with respiratory complications and cardiomyopathy being common causes of death. BMD is a milder form
with a later onset and a much longer survival. In BMD
patients, wheelchair dependency typically occurs after age 16
(Bushby et al. 2010).
∗ For correspondence. E-mail: [email protected].
Both DMD and BMD are caused by mutations in the dystrophin gene which spans 2.4 Mb at Xp21.2 (Den Dunnen et al.
1989; Walmsley et al. 2010). Dystrophin protein is found
at the inner surface of muscle fibre and anchors muscle cell
membrane to the extracellular matrix (Campbell 1995). The
extreme large dystrophin gene size accounts for a high mutational rate. Approximately two-thirds of the gene mutations
are large deletions and duplications, and the remaining onethird are smaller mutations such as nucleotide substitution or
small indels (Roberts et al. 1992). Generally, DMD patients
harbour nonsense or frameshift mutations which cause premature translation termination, whereas BMD patients carry
in-frame mutations which result in reduced molecular weight
or expression level. Therefore, the severity of the disease is
determined by whether the mutation maintains an open reading frame (Monaco et al. 1988). Typically, large deletions
cluster in two hotspot regions of the gene, but small deletions and point mutations appear to have even distribution,
although more than 40% of the small mutations are found
Keywords. Duchenne and Becker muscular dystrophy; neuromuscular disorder; point mutation.
Journal of Genetics, Vol. 95, No. 2, June 2016
325
Maryam Haghshenas et al.
3 to exon 55 (Prior et al. 1995; Lalic et al. 2005). Point
mutations including nonsense, missense and splice site mutations are more difficult to detect because of the enormous size
of the gene (Rininsland and Reiss 1994).
In this study, a total of 29 DNA samples from patients
were sequenced for detection of point mutations in exons 50–
79. The investigation for DMD point mutations as far as the
authors are aware has not taken place on Iranian patients and
this report is the first in this respect.
Materials and methods
Twenty-nine unrelated male patients (17 DMD and 12 BMD)
from different regions of Iran, who were tested negative
for deletions/duplicatons in dystrophin gene (KhordadpoorDeilamani et al. 2011), were subjected to point mutation
analysis for exons 50–79. Patients’ mean age for DMD and
BMD was 7.49 and 21.58, respectively. Diagnosis was based
on EMG and cardinal clinical signs of the disease. Informed
written consent was obtained from all patients or their parents
prior to analysis. Genomic DNA was extracted from peripheral whole blood of the patients and their parents (when
available) using salting out procedure. The primers were
designed using gene runner software (table 1).
Exons 50–79 in all patients were amplified by PCR. Also
exon 44 was amplified in one patient (N5). In this patient
MLPA result indicated a probable deletion for exon 44,
although multiplex and single PCR testing showed that there
was no deletion. Because of this controversy, this patient was
also included in the study. PCR amplification was performed
at a total volume of 30 μL containing 100 ng genomic DNA,
1× PCR buffer, 1.5 mM MgCl2 , 0.2 mM dNTP, 0.5 μM each
primer and 1.5 units of Taq DNA polymerase (CinnaGen,
Iran). PCR condition was as follows: an initial denaturation
step for 5 min at 95◦ C, followed by 30 cycles of denaturation for 45 s at 95◦ C, annealing for 45 s according to temperatures described in table 1, extension for 45 s at 72◦ C, and
a final extension for 10 min at 72◦ C. Cycle sequencing was
performed by Macrogen, South Korea.
Results
Sequence variations were observed in nine patients, including four nonsense, one frame shift and two splice site
mutations as well as one missense polymorphism and one
missense variant with unknown significance (table 2). Nonsense mutations causing stop codon were identified at exons
57, 58, 62 and 66 (patients N1–N4). An insertion of
nucleotide T at the position c.6380-81 (exon 44) resulted in a
frameshift in the reading frame which later led to the formation of premature stop codon at six amino acids afterwards
(patient N5). The patients N6 and N7 showed nucleotide
substitution of G to C in intron 62 and G to A in intron
65, respectively, causing splice-site mutations. Polymorphic
variant was detected in patient N8. Nucleotide substitution of
326
G to A which resulted in amino acid change from serine to
asparagine in exon 54 was observed in patient N9 (figure 1).
Discussion
This is the first molecular analysis of dystrophin gene
applying direct sequencing to detect point mutations in nondeletion/duplication DMD/BMD patients in Iran. Use of molecular techniques has made the unpopular invasive techniques
such as diagnostic muscular biopsy redundant and unnecessary to a great extent. Considering the sizeable occurrence of
the point mutations in dystrophin gene, more investigation
should be carried out in patients when MLPA could not identify any mutation. Owing to enormous size of the dystrophin
gene and widespread dispersal of point mutations within this
gene, investigation of all 79 exons is difficult, expensive and
labour-intensive. As it is known, about 40% of the DMD
point mutations occur in 3 -end of the dystrophin gene (Prior
et al. 1995), therefore, we focussed on the last 30 exons, from
50 to 79 (about 38% of the exons). In the present study, we
aimed at identifying point mutations in 29 unrelated patients
with no deletion/duplication mutations. Nonsense mutations
(in exons 57, 58, 62 and 66) were identified in four patients
(N1–N4) who had been diagnosed in early life and symptoms
correlated with severe form of the disease (DMD phenotype).
The other patients who are reported to have nonsense mutations, also exhibited severe Duchenne phenotype (Schwartz
and Duno 2004; Taylor et al. 2007; Flanigan et al. 2009;
Sedlackova et al. 2009; Takeshima et al. 2010; Chen et al.
2013). Patient N5 was already screened by MLPA method
which showed the deletion of exon 44, while single and
multiplex PCR revealed that the detected deletion was not
genuine. Subsequent sequencing revealed that a novel point
mutation has occurred within that exon near the ligation site
of the MLPA probes. An insertion of nucleotide T at the position c.6380-81 resulted in a frameshift in the reading frame
and later led to the formation of premature stop codon at six
amino acids afterwards. In patient N6, the splice site mutation in intron 62, obliterated the splicing site and resulted in
a frameshift in the reading frame, and it was known to cause
DMD rather than BMD and our patient demonstrated DMD
phenotype (Gardner et al. 1995). Another splice site mutation detected in patient N7 was a nucleotide substitution of
G to A in splice donor recognition site which led to the use
of a cryptic splice site, with consequent insertion of intronic
sequences into the processed mRNA. This novel mutation
lies within the region coding for the CR domain of dystrophin
that is important for establishing a series of important interactions with beta-dystroglycan (Vulin et al. 2014). Different from what observed in patient 6 (in which the frameshift
should abolish the entire CR domain), this mutation found
in patient 7 should at least preserve the WW domain. The
patient’s mother who was also screened for this mutation
reveled that she had this mutation. In patient N8, nucleotide
substitution (A8762G) in exon 59 resulted in an amino acid
Journal of Genetics, Vol. 95, No. 2, June 2016
Point mutations in dystrophin gene
Table 1. PCR primer sequences for exons 44 and 50–79 of dystrophin gene.
Exon
Exon 44
Exon 50
Exon 51
Exon 52
Exon 53
Exon 54
Exon 55
Exon 56
Exon 57
Exon 58
Exon 59
Exon 60
Exon 61
Exon 62
Exon 63
Exon 64
Exon 65
Exon 66
Exon 67
Exon 68
Exon 69
Exon 70, 71
Exon 72
Exon 73
Exon 74
Exon 75
Exon 76
Exon 77
Exon 78
Exon 79
Forward primer (5 →3 )
Reverse primer (5 →3 )
PCR product (bp)
Annealing temperature
268
59
271
61
388
64
468
58
592
60
450
64
408
56
353
62.5
320
61
280
60
645
61
523
58
523
58
523
60
523
55
523
55
400
58
380
57
400
55
400
57
324
55
1114
61
547
57
547
56
455
58
520
58
443
58
474
56
474
57
600
59
CTTGATCCATATGCTTTTACCTGCA
TCCATCACCCTTCAGAACCTGACCT
CACCAAATGGATTAAGATGTTCATGAAT
TCTCTCTCACCCAGTCATCACTTCATAG
GAAATTGGCTCTTTAGCTTGTGTTTC
GGAGAGTAAAGTGATTGGTGGAAAATC
CTTACTCAAGGCATTCAGACAGTG
TGAAACTTGTCATGCATCTTGC
AATCCTGTTGTTCATCATCCTAGC
CCCAGAGTTCAAGGCTCAGTG
GTTTGTCCTGAAAGGTGGGTTAC
TTATCGTCTTGAACCCTCCCAAG
AATTTAGTTCCTCCATCTTTCTCT
AAATACATCAGGCTGTATAAAAGC
ATTCTGCACATATTCTTCTTCCTGC
GGATGATTTACGTAGACATGTGAG
CAATGGAATTGTTAGAATCATCA
CACTGGATTACTATGTGCTTAACAT
TTTTGAGAAGAATGCCACAAGCC
AAAATATGAGAGCTATCCAGACC
CTCTTTCTTTCTTCCAGTATGACC
ACACACACACACACACATCAGAC
CACTGCACCCTAAAGAGAATAAGC
TGAGCCAAATAAACACCTGTTC
TTGTGTTTGGCCTTGGATTG
AGAATACTAGCATGGCTCCTGAG
CTGTCTTCATGGGCAGCTGAG
AGGTATTGTAGGCCAGGCTAATG
TCATTCAGGCCCAAGTAAATATAG
TTCACACTGCAAACTACTTTATCC
TTTGTGGTGGTTGTAGTTATTGG
TCAACATCAGTAGCTGAGGAATGG
CACCTTTCAAAGAGAGTGTGGTTC
TGTACGCTAAGCCTCCTGTGAC
TTCACACATCATTGAGCACTTTAC
AATGTTTGACAAGGAATGGCAC
CACTCTGTGGAAATACTGGCTAC
CAAATCCCATACCTACTGCCTAC
TTTGGATATCAGGGAGCATTTG
ACTAACAGCAACTGGCACAGG
AAATCTGGAGGCAGAAAGTTAATC
ACTGAAATTTATCCCAGGTGAAC
TCAGCCAAATTAGTACATGCCAG
AAGCGAGCGAATGTGTTGGTG
TGCTTCCTAGAAACTGTCTCCC
ACTAATGCTCTTGTTGGTGACC
TCTCTTCTTGCCATGATTTATCC
ATGCTATACCAAGATCACGTTTCC
TTCAATTGTCAGCTGTAGAATGAG
TAGATTTCTGAAGCCAACCACAC
CTGTTCTTCGGTGGCAGTCAG
CCTTTGATACTATCACTTTGCAGG
TGGGAGGGCTTCTAAAGTAGG
CTGTGGGCCAGATAGAGCTG
TGACAGAGCAAGACTCCATCTC
CATTCTGATACTGCGTGTTGG
TTTACAAATGACGTCTTCTGGATC
CTGCAAGTGGAGAGGTGACTAC
GCAGCTTCTGTGTTGTCTTCAC
TGTATGTGTGTGTGTGTGTGTTTG
Journal of Genetics, Vol. 95, No. 2, June 2016
327
Maryam Haghshenas et al.
Table 2. Spectrum of dystrophin gene mutations detected in the present study.
Patient
N1
N2
N3
N4
N5∗
N6
N7∗
N8
N9∗
Age of
diagnosis
Phenotype
Location
Systematic name
(DNA level)
Trivial name
( protein effect)
6
8
8
4
5
5
7
10
25
DMD
DMD
DMD
DMD
DMD
DMD
DMD
DMD
BMD
Exon 57
Exon 58
Exon 62
Exon 66
Exon 44
Intron 62
Intron 65
Exon 59
Exon 54
c. (8460 G>A)
c. (8608 C>T)
c. (9182 G>A)
c. (9568 C>T)
c. (6380-81 ins T)
c. (9224+5 G>C)
c. (9563+1 G>A)
c. (8762 A>G)
c. (8015 G>A)
p.Trp 2820 X
p.Arg 2870 X
p.Trp 3061 X
p.Arg 3190 X
p.2127ins T FsX6
IVS62+5
IVS65+1
p.His 2921 Arg
p.Ser 2672 Asn
Characters of mutation
Nonsense
Nonsense
Nonsense
Nonsense
Frameshift
Splicing
Splicing
Missense polymorphism
Missense (with unknown significance
probably a benign variant)
*Novel variants.
Figure 1. Automated DNA sequencing electropherogram. Arrows indicate the nucleotide substitution c. (8460 G>A)
in exon 57 (N1), c. (8608 C>T) in exon 58 (N2), c. (9182 G>A) in exon 62 (N3), c. (9568 C>T) in exon 66 (N4), c.
(6380-81 ins T) in exon 44 (N5), c. (9224+5 G>C) in intron 62 (N6), c. (9563+1 G>A) in intron 65 (N7), c. (8762
A>G) in exon 59 (N8) and c. (8015 G>A) in exon 54 (N9).
alteration from histidine to arginine at position 2921 of dystrophin protein. This change probably does not affect function and was considered as polymorphism (Hofstra et al.
1994, Nigro et al. 1994, Almomani et al. 2009). Nucleotide
substitution of G to A (G8015A) in exon 54 of patient N9
led to change of amino acid serine to asparagine. The murine
dystrophin harbours an asparagine residue within the same
topological position (2665 in mouse). Thus, Ser2672Asn is
likely to represent a variant producing a very mild condition. Examination of this mutation by SIFT software
revealed that this variant is not pathogenic (http://sift.jcvi.
org/). The patient’s age at referral was 25 years, and the
clinical symptoms were mild and age of onset was after
puberty.
328
In conclusion, by scanning less than 40% of dystrophin
exons in 29 DMD/BMD patients, seven point mutations as
well as two benign variants were identified. As far as 20
unidentified patients are concerned, they may carry mutations located upstream of exon 50 which have not been
screened in this study. Certainly, the genetic determinants of
some of the patients may reside in other genes resulting in
muscular dystrophies overlapping in phenotype with DMD.
The data generated in the present study and similar studies in addition to increasing our understanding of structure
and function of dystrophin gene and their correlation with
the disease could be utilized in the context of carrier detection testing of females in the patients’ families and prenatal
diagnosis.
Journal of Genetics, Vol. 95, No. 2, June 2016
Point mutations in dystrophin gene
Acknowledgements
We thank all the participants who assisted in this survey. This study
was supported by Tehran Medical Genetics Laboratory, Tehran, Iran
(grant number 90002).
References
Almomani R., van der Stoep N., Bakker E., den Dunnen J. T.,
Breuning, M. H. and Ginjaar I. B. 2009 Rapid and cost effective
detection of small mutations in the DMD gene by high resolution
melting curve analysis. Neuromuscul. Disord. 19, 383–390.
Bushby K., Finkel R., Birnkrant D. J., Case L. E., Clemens P. R.,
Cripe L. et al. 2010 Diagnosis and management of Duchenne
muscular dystrophy, part 2: implementation of multidisciplinary
care. Lancet Neurol. 9, 177–189.
Campbell K. P. 1995 Three muscular dystrophies: loss of
cytoskeleton-extracellular matrix linkage. Cell 80, 675–679.
Chen W. J., Lin Q. F., Zhang Q. J., He J., Liu X. Y., Lin M. T. et al.
2013 Molecular analysis of the dystrophin gene in 407 Chinese
patients with Duchenne/Becker muscular dystrophy by the combination of multiplex ligation-dependent probe amplification and
Sanger sequencing. Clin. Chim. Acta 423, 35–38.
Den Dunnen J. T., Grootscholten P. M., Bakker E., Blonden L. A.,
Ginjaar H. B., Wapenaar M. C. et al. 1989 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. 45, 835–847.
Flanigan K. M., Dunn D. M., von Niederhausern A., Soltanzadeh
P., Gappmaier E., Howard M. T. et al. 2009 Mutational spectrum
of DMD mutations in dystrophinopathy patients: application of
modern diagnostic techniques to a large cohort. Hum. Mutat. 30,
1657–1666.
Flanigan K. M., Dunn D. M., von Niederhausern A., Soltanzadeh
P., Howard M. T., Sampson J. B. et al. 2011 Nonsense mutationassociated Becker muscular dystrophy: interplay between exon
definition and splicing regulatory elements within the DMD gene.
Hum. Mutat. 32, 299–308.
Gardner R. J., Bobrow M. and Roberts R. G. 1995 The identification
of point mutations in Duchenne muscular dystrophy patients by
using reverse-transcription PCR and the protein truncation test.
Am. J. Hum. Genet. 57, 311–320.
Hegde M. R., Chin E. L., Mulle J. G., Okou D. T., Warren S. T. and
Zwick M. E. 2008 Microarray-based mutation detection in the
dystrophin gene. Hum. Mutat. 29, 1091–1099.
Hofstra R. M., Mulder I. M., Vossen R, de Koning-Gans P. A.,
Kraak M., Ginjaar I. B. et al. 2004 DGGE-based whole-gene
mutation scanning of the dystrophin gene in Duchenne and
Becker muscular dystrophy patients. Hum. Mutat. 23, 57–66.
Khordadpoor-Deilamani F., Akbari M. T., Nafissi S. and Zamani
G. 2011 Dystrophin gene mutation analysis in Iranian males and
females using multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification methods. Genet. Test
Mol. Biomarkers 15, 893–899.
Lalic T., Vossen R. H., Coffa J., Schouten J. P., Guc-Scekic M.,
Radivojevic D. et al. 2005 Deletion and duplication screening in
the DMD gene using MLPA. Eur. J. Hum. Genet. 13, 1231–1234.
Monaco A. P., Bertelson C. J., Liechti-Gallati S., Moser H. and
Kunkel L. M. 1988 An explanation for the phenotypic differences
between patients bearing partial deletions of the DMD locus.
Genomics 2, 90–95.
Nigro V., Nigro G., Esposito M. G., Comi L. I., Molinari A.
M., Puca G. A. et al. 1994 Novel small mutations along the
DMD/BMD gene associated with different phenotypes. Hum.
Mol. Genet. 3, 1907–1908.
Prior T. W., Bartolo C., Pearl D. K., Papp A. C., Snyder P. J., Sedra
M. S. et al. 1995 Spectrum of small mutations in the dystrophin
coding region. Am. J. Hum. Genet. 57, 22–33.
Rininsland F. and Reiss J. 1994 Microlesions and polymorphisms
in the Duchenne/Becker muscular dystrophy gene. Hum. Genet.
94, 111–116.
Roberts R. G., Bobrow M. and Bentley D. R. 1992 Point mutations
in the dystrophin gene. Proc. Natl. Acad. Sci. USA 89, 2331–
2335.
Schwartz M. and Duno M. 2004 Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligationdependent probe amplification method. Genet. Test 8, 361–
367.
Sedlackova J., Vondracek P., Hermanova M., Zamecnik J., Hruba
Z., Haberlova J. et al. 2009 Point mutations in Czech DMD/BMD
patients and their phenotypic outcome. Neuromuscul. Disord. 19,
749–753.
Takeshima Y., Yagi M., Okizuka Y., Awano H., Zhang Z., Yamauchi
Y. et al. 2010 Mutation spectrum of the dystrophin gene in 442
Duchenne/Becker muscular dystrophy cases from one Japanese
referral center. J. Hum. Genet. 55, 379–388.
Taylor P. J., Maroulis S., Mullan G. L., Pedersen R. L., Baumli A.,
Elakis G. et al. 2007 Measurement of the clinical utility of a combined mutation detection protocol in carriers of Duchenne and
Becker muscular dystrophy. J. Med. Genet. 44, 368–372.
Vulin A., Wein N., Strandjord D. M., Johnson E. K., Findlay A.
R., Maiti B. et al. 2014 The ZZ domain of dystrophin in DMD:
making sense of missense mutations. Hum. Mutat. 35, 257–264.
Walmsley G. L., Arechavala-Gomeza V., Fernandez-Fuente M.,
Burke M. M., Nagel N., Holder A. et al. 2010 A Duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient
cavalier king charles spaniels is amenable to exon 51 skipping.
PLoS One 5, e8647.
Received 2 July 2015, in revised form 29 August 2015; accepted 6 October 2015
Unedited version published online: 9 October 2015
Final version published online: 16 May 2016
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