Molecular deletion patterns in Duchenne and Becker muscular dystrophy patients from KwaZulu Natal.

Abstract

There exists much phenotypic heterogeneity in Duchenne muscular dystrophy and its allelic variant, Becker muscular dystrophy. The molecular findings on 53 patients with Duchenne and 15 patients with Becker type muscular dystrophy in KwaZulu Natal, South Africa are reported. Multiplex PCR was performed using primers targeting 18 hot-spot exons throughout the dystrophin gene. Analysis of the multiplex PCR data revealed that 39/68 (57.0%) patients included in the study showed a deletion (33 DMD and 6 BMD patients). Twenty-five patients were Black, 4 were White and 10 were Indian. Using the Chamberlain and Beggs multiplex PCR assays, the region of the genome most frequently affected by a deletion includes exons 47-51. The distal region of the dystrophin gene was most frequently affected by the deletion in both Black and Indian patients. There were too few White patients for conclusions to be drawn concerning the most frequently affected part of the gene. Although the numbers are insufficient to determine whether ethnic differences are present, the Chamberlain and Beggs multiplex PCR assays detect deletions with the same frequency in South African DMD/BMD patients as that reported in the literature.

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Molecular deletion patterns in Duchenne and Becker muscular
dystrophy patients from KwaZulu Natal
K.D. Hallwirth Pillaya, P.L.A. Billb,⁎, S. Maduraia, L. Mubaiwac, P. Rapitic
aNeuroscience Laboratory, Department of Neurology, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa
bDepartment of Neurology, IALCH and Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa
cDepartment of Paediatric Neurology, IALCH and Nelson R Mandela School of Medicine, University of KwaZulu-Natal, South Africa
Received 14 October 2005; received in revised form 29 June 2006; accepted 30 June 2006
Available online 1 December 2006
Abstract
There exists much phenotypic heterogeneity in Duchenne muscular dystrophy and its allelic variant, Becker muscular dystrophy. The
molecular findings on 53 patients with Duchenne and 15 patients with Becker type muscular dystrophy in KwaZulu Natal, South Africa are
reported. Multiplex PCR was performed using primers targeting 18 hot-spot exons throughout the dystrophin gene. Analysis of the multiplex
PCR data revealed that 39/68 (57.0%) patients included in the study showed a deletion (33 DMD and 6 BMD patients). Twenty-five patients
were Black, 4 were White and 10 were Indian. Using the Chamberlain and Beggs multiplex PCR assays, the region of the genome most
frequently affected by a deletion includes exons 47–51. The distal region of the dystrophin gene was most frequently affected by the deletion
in both Black and Indian patients. There were too few White patients for conclusions to be drawn concerning the most frequently affected
part of the gene. Although the numbers are insufficient to determine whether ethnic differences are present, the Chamberlain and Beggs
multiplex PCR assays detect deletions with the same frequency in South African DMD/BMD patients as that reported in the literature.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Duchenne muscular dystrophy; Becker muscular dystrophy; Dystrophin deletions; Polymerase chain reaction; Molecular biology; Genetics
1. Introduction
Duchenne muscular dystrophy is an X-linked disorder
with a reported incidence of 1:3500 live male births in
European children [1]. Signs usually become evident
between the ages 3–5 years. In Becker muscular dystrophy
(BMD), the allelic variant of DMD, the clinical course is less
severe and onset is later at about 11 years with the mean age
of wheelchair confinement of approximately 27 years [1].
The dystrophin gene is approximately 2.3 Mb and
contains 79 exons encoding a 14 Kb mRNA. The 427 kDa
dystrophin protein contains 3685 amino acids [2]. Large
deletions and duplications are found in approximately 65%
of patients [3] and are described as occurring predominantly
in two high frequency deletion regions (HFDR) termed hot-
spots. According to Beggs et al. [4], 98% of deletions are
detected using primers targeting 18 hot-spot exons in the
dystrophin gene. The proximal hot-spot encompasses exons
3–7 and the distal hot-spot exons 45–51. There is little
information concerning patterns and frequency of deletions
in DMD/BMD patients in South Africa. One previous study
in South Africa documented deletions in 42% of patients
studied and only 26% of Black patients in this group were
found to have a deletion [5]. There is little information on the
racial distribution of deletions in DMD in the literature. The
aim of this study is to identify and characterise the deletion
patterns in DMD/BMD patients of different racial groups in
KwaZulu Natal, South Africa using primers targeting 18 hot-
spot exons in the dystrophin gene.
2. Materials and methods
Patient population: Sixty-eight patients with a dystro-
phinopathy (DMD or BMD) were studied. Fifteen of the
Journal of the Neurological Sciences 252 (2007) 1–3
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⁎Corresponding author. Department of Neurology, Inkosi Albert Luthuli
Hospital, Private Bag X03, Mayville, 4058, Durban, South Africa, Tel.: +27
31 240 2359/62; fax: +27 31 240 2358.
0022-510X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jns.2006.06.025

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68 patients had features consistent with Becker muscular
dystrophy. The remaining 53 patients were diagnosed as
DMD. Forty of the 68 patients were Black (29 DMD, 11
BMD), 19 were Indian (17 DMD, 2 BMD) and 9 were
White (7 DMD, 2 BMD). The age range of BMD patients
was 10–23 years and 3–26 years for DMD patients. The
patients were diagnosed on the basis of proximal weakness
with onset in childhood, a selective pattern of muscle
weakness in the pelvic and shoulder girdle muscles,
markedly elevated CK levels and male sex. Patients were
considered to have BMD if onset of weakness was after
the age of 10 years or if the patients were ambulant up to
the age of 16 years. Twelve of the 68 patients showed
varying degrees of mental retardation. Forty-four patients
underwent muscle biopsy with informed consent. The
biopsies showed features typical of a dystrophic pattern
and absence of dystrophin on immunoperoxidase staining
in the DMD patients and reduced staining in BMD
patients. The patients were all unrelated. Ethics approval
was obtained from the Bioethics Committee of the
University of KwaZulu-Natal (Ref. H021/01).
DNA Isolation: Blood samples were obtained from
patients after written informed consent was obtained. DNA
was extracted from whole blood using the QIAamp DNA
blood mini kit (Qiagen, 1999).
PCR assay and electrophoresis: For the PCR assay the
Amplitaq DNA polymerase system was used according to
the methodology recommended by Applied Biosystems.
The Chamberlain and Beggs set of primers were used.
The following amendments were made to the PCR
conditions: For the Chamberlain set, a hot start was
included at 95 °C for 1–2 min, whilst the DNA was
being added to the mixture. The initial denaturation was
performed at 95 °C for 5 min. The denaturation during
the cycling was performed at 94 °C for 30 s. The final
elongation cycle was carried out at 65 °C for 5 min
instead of the recommended 7 min in the paper. The only
change made to the PCR conditions using the Beggs set
of Primers was an increase in cycle number from 25 to
32. The amplified products were electrophoresed on a
2.5% Nusieve agarose gel. The 68 patients included in
the study were categorised into deletion and non-deletion
groups.
3. Results
A deletion was found in 39/68 patients studied (57%),
(62% of black patients, 52% of Indian patients and 44% of
white patients), as shown in Table 1. With the Chamberlain
and Beggs assay, the most frequently affected region
encompassed exons 47–51. In the 19/29 Black DMD
patients with a deletion, 1 had a deletion spanning the
proximal part of the gene, 11 had a deletion encompassing
the distal part of the gene, and 7 had a deletion encompassing
both the proximal and distal parts of the gene. 6/11 black
BMD patients had a deletion affecting the distal (3) or
proximal (3) part of the gene. In 10/17 Indian DMD patients
with a deletion, 2 had a deletion spanning the proximal part
of the gene and 8 had a deletion encompassing the distal part
of the gene. Of the 4/9 White DMD patients with a deletion,
1 had a deletion encompassing the proximal part of the gene,
1 had a deletion encompassing the distal part of the gene, and
2 had a deletion spanning both the proximal and distal part of
the gene. None of the 4 Indian and White BMD patients
showed a deletion.
Twelve of the 68 (17.6%) patients showed varying
degrees of mental retardation (MR). Four of these patients
showed a deletion. Three of the 4 patients were DMD
patients; 1 had a deletion in the proximal part of the gene, 1
showed a deletion in the distal part of the gene and 1 had a
deletion encompassing the distal and proximal part of the
gene. The 1 patient with a BMD phenotype and MR had a
small deletion in the distal part of the gene. The other 4
(33%) patients had no deletions.
4. Discussion
In the present study, 18 exons were screened. Therefore a
complete representation of the size of the deletion in the
dystrophin gene cannot be determined. However, literature
consensus has shown that the screening of 18 hot-spot exons
detects 98% of deletions(4). Deletions in our patients were
clustered in the high frequency deletion regions (HFDR),
located at the 5' proximal end of the gene, with deletions
spanning exons 3–7, and at the distal hot-spot encompassing
deletions of exons 45–51 as shown in Table 1. In this study,
the percentage of Black and Indian DMD/BMD patients with
Table 1
Analysis of data for exons found to be deleted using 18 exon specific primer pairs in the Chamberlain and Beggs sets
Ethnic groupNo. of
patients
Mental
retardation
No. of patients
with deletions
Deletion site
ProximalDistalProximal+Distal
Black DMD
BMD
Indian DMD
BMD
White DMD
BMD
29
11
17
2
7
2
3
1
0
0
0
0
19
6
10
0
4
0
1
0
2
–
1
–
11
3
8
–
1
–
7
3
0
–
2
–
DMD: Duchenne muscular dystrophy.
BMD: Becker muscular dystrophy.
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deletionsissimilartothatreportedintheliterature.Duetothe
small number of white patients it is not possible to comment
on the lower incidence of deletions in this group. In the only
previouslyreportedstudyofSouthAfrican patients[5],Ballo
et al. found deletions in 26% of the 43 black DMD patients,
59% of 23 White DMD patients, 45% of 21 Indian DMD
patients and 43% of 27 mixed ancestry DMD patients. In our
studythepercentageofblackpatientswithadeletion(63%)is
similar to that reported in the literature and does not support a
lower incidence of deletions in Black patients.
In Chinese patients, Zhang [6] reported deletions in 31/60
(52%) DMD/BMD patients and Soong et al. [7] found
deletions in 13/29 (45%) of patients. These were clustered
primarily near the central part of the gene. Katayama et al.
[8] studied 27 Japanese DMD families and detected deletions
by PCR in 52% and by Southern blot in 64% of the families.
Hiyama et al. [9] found deletions in 54% and partial
duplications in 10% of 59 Japanese patients with DMD.
Kitoh et al. [10] studied 99 Japanese patients with DMD
and found deletions in 40% of patients. In the West Midlands
region of Britain, Roddie and Bundey [11] reported that
DMD was twice as common as expected in Indians and less
common than expected in Pakistanis.
When comparing the site of the deletion in the different
race groups in our study, none of the Indian patients had a
deletion encompassing both the proximal and distal part of
the gene, whereas in Blackpatients 7/19 of the DMD patients
and 3/6 of the BMD patients had a deletion encompassing
both the proximal and distal part of the gene. In Black and
Indian patients, a deletion was more common in the distal
part of the gene compared to the proximal part. There were
too few White patients for comparison with the other racial
groups, and overall there are insufficient numbers of patients
for statistical analysis to be performed
The multiplex PCR for deletion detection serves as an
essential tool for diagnosis of DMD/BMD. A disadvantage
of using this PCR amplification method for deletion
detection is that duplications and aberrant sized junction
fragments and point mutations will not be detected. In
conclusion this study has shown that overall 57% (39/68) of
clinically manifesting DMD/BMD patients were found to
have a deletion when multiplex PCR was used to screen the
18 hot-spot exons throughout the dystrophin gene, and that
black DMD/BMD patients have a similar frequency of
deletions as that reported elsewhere.
Acknowledgements
The authors would like to acknowledge Professor Bhigjee
for his valuable input in reading the article, and The
Muscular Dystrophy Foundation, the National Research
Foundation and the University of Natal Research Fund for
funding the project.
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