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ORIGINAL ARTICLE
Year : 2019  |  Volume : 67  |  Issue : 3  |  Page : 714-715

The spectrum of deletions and duplications in the dystrophin (DMD) gene in a cohort of patients with Duchenne muscular dystrophy in Sri Lanka


Human Genetics Unit, Faculty of Medicine, University of Colombo, Sri Lanka

Date of Web Publication23-Jul-2019

Correspondence Address:
Dr. Vajira H W Dissanayake
Human Genetics Unit, Faculty of Medicine, University of Colombo, Kynsey Road, Colombo 8
Sri Lanka
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.263235

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 » Abstract 


Background: Duchenne muscular dystrophy (DMD), which affects 1 in 3500 newborn males, is the most common fatal neurodegenerative disorder in children. Deletions and duplications in the DMD gene are the most common underlying etiological factors.
Materials and Methods: Fifty consecutive children with DMD were screened for deletions and duplications in the DMD gene using Multiple Ligation-binding Probe Amplification (MLPA).
Results: Forty (80%) children had deletions and 4 (8%) had duplications. Single exon involvement was seen in 8 (16%), two exon involvement was seen in 3 (6%), three exon involvement was seen in 6 (12%) children, and four exon involvement in 1 (2%) child. More than four exon involvement were seen in 26 (52%) children. The most common deletion was the deletion spanning from exon 45 to exon 52, which was seen in 6 (12%) children. The next common exon deletion was single exon 45 deletion seen in 4 (8%) children. The most frequent mutant region fell within exons 45 to 55 (52%) followed by within exons 21 to 44 (26%) and exons 1 to 20 (26%). The least common region fell within exons 56 to 79 (4%).
Conclusion: The deletion/duplication pattern seen in this cohort of children with DMD was similar to that reported among other global populations.


Keywords: Deletions, Duchenne muscular dystrophy, duplications, multiple ligation binding probe amplification, Sri Lanka
Key Message: This paper for the first time documents the complete spectrum of deletions seen in patients with DMD in the Sri Lankan population.


How to cite this article:
Thakur N, Abeysekera G, Wanigasinghe J, W Dissanayake VH. The spectrum of deletions and duplications in the dystrophin (DMD) gene in a cohort of patients with Duchenne muscular dystrophy in Sri Lanka. Neurol India 2019;67:714-5

How to cite this URL:
Thakur N, Abeysekera G, Wanigasinghe J, W Dissanayake VH. The spectrum of deletions and duplications in the dystrophin (DMD) gene in a cohort of patients with Duchenne muscular dystrophy in Sri Lanka. Neurol India [serial online] 2019 [cited 2019 Aug 19];67:714-5. Available from: http://www.neurologyindia.com/text.asp?2019/67/3/714/263235




Duchenne muscular dystrophy (DMD) is a rapidly progressive X-linked recessive neuromuscular disorder with a worldwide incidence of 1/3500 among male live births.[1] It is one of the most common inherited neuromuscular disorders seen in the pediatric age group. It is caused by mutations in the DMD gene, one of the largest known genes in human genome with 79 exons and 8 tissue-specific promoters encoding a 14-kb messenger ribonucleic acid (mRNA).[2] Being the largest gene described, spanning 2.4 Mb, the DMD gene is prone to mutation. Mutations leading to a truncated protein cause the severe phenotype of DMD, whereas mutations retaining the mRNA reading frame cause the relatively milder phenotype of Becker muscular dystrophy (BMD) with an incidence of 1/18000.[3],[4] The DMD gene codes for the dystrophin protein, the levels of which are very low (<3% of normal) or absent in DMD; however, patients with BMD have 10–40% of the normal amount of dystrophin but with an abnormal molecular weight. Dystrophin is expressed in a variety of tissues including skeletal muscles, heart, and brain. Therefore, deficiency of dystrophin causes widespread clinical manifestations of the disease.[5] Testing for mutations in large genes, such as in the DMD gene, has been a challenging venture until recently.[6] Deletions, duplications, and point mutations have been reported in most exons of the DMD gene. Deletions account for approximately 65% of the mutations, whereas duplications account for 6%, and point mutation for the rest. Although they are very heterogeneous, the majority of large deletions cluster around two mutation hotspot regions. The first deletion cluster region spans from exons 44 to 53, whereas the second one spans from exons 2 to 20.[7] Until recently it was not possible to screen all 79 exons of the DMD gene for deletions and duplications in a cost-effective manner. The introduction of the multiplex ligation probe amplification (MLPA) technique has made this possible.[8] The objective of this study was to describe the deletion/duplication profile of a cohort of patients with DMD in the Sri Lankan population and compare it with that reported in other populations.


 » Materials and Methods Top


Fifty children, clinically diagnosed with DMD, who were referred to the Human Genetics Unit, Faculty of Medicine, University of Colombo, were tested for mutations in the DMD gene after obtaining written informed consent from their parents or guardians. The study was performed according to a protocol approved by the Ethics Review Committee of the Faculty of Medicine, University of Colombo. The clinical details including a three-generation pedigree were recorded.

Genotyping

All individuals were tested for deletions and duplications in the DMD gene using the SLASA MLPA 095 kit (MRC Holland, Netherlands) according to the manufacturer's protocol. MLPA reactions were carried out on an Applied Biosystems 2720 Thermal Cycler. Capillary electrophoresis and fragment analysis was done on an Applied Biosystems 3130 DNA analyzer. Data were analyzed as recommended by the manufacturer [Figure 1].
Figure 1: Mutation pattern in the DMD gene. From top to bottom on left column represents the number of patients (REF 1-50) and from left to right on the first row represents the number of exons (1-79) in the DMD gene. The grey shaded area shows duplications whereas the black shaded area shows deletions in the DMD gene

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 » Results Top


Clinical phenotype characterization

As DMD is an X-linked recessive disorder, a three-generation pedigree was drawn for every child. Fourteen (28%) children had a family history of DMD. In 13 of these families with a family history of DMD, the proband had an affected sibling.

Molecular genetic testing

Deletions were found in 40 (80%) children and duplications were found in 4 (8%). Deletions or duplications were not detected in 6 (12%) children. When exons involved in deletions or duplications were examined, single exon involvement was seen in 8 (16%) children, two exons were seen in 3 (6%), three exons were seen in 6 (12%), four exons were seen in 1 (2%), and more than four exonal involvement was seen in 26 (52%) children [Figure 2].
Figure 2: MLPA output showing deletions from exon 45 to exon 50 in the DMD gene in a child with DMD

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The most common deletion was an eight-exon deletion ranging from exon 45 to exon 52, which was seen in 6 (12%) children, as shown in [Figure 1]. The next common deletion was a single exon deletion involving exon 45, which was seen in 4 (8%) children.

The 79 exons of the dystrophin gene were divided into four groups. The first group ranged from exon 1 to exon 20, the second group ranged from exon 21 to 44, the third group ranged from exon 45 to 55, and the fourth group ranged from exon 56 to 79. In this study, the most frequent mutant region fell within exons 45 to 55 [26 (52%)], followed by exons 21 to 44 [13 (26%)], and exons 1 to 20 [13 (26%)]. The least common region fell within exons 56 to 79 (4%), as shown in [Figure 1].


 » Discussion Top


We applied the MLPA technique for the first time in Sri Lanka as a diagnostic tool to detect deletions and duplications in the DMD gene. The diagnosis was confirmed in 44 out of 50 children (88%). The diagnostic yield of detecting DNA rearrangements has been increased by 25.5% compared to a previous study in Sri Lanka (15 out of 24; 62.5%) using the multiplex polymerase chain reaction (PCR) method.[9] The deletion frequency of the DMD gene in Sri Lanka (80%) was comparable to North, South, East and West Indian populations; however, they were higher than most of the other Asian countries.[10] According to a systematic review conducted in 2016, the prevalence of mutations of the DMD gene is approximately 68%.[11] We could not compare the duplication frequency as our data was not sufficient, and we suggest further studies should be carried out to assess duplications in a larger sample. The above mentioned systematic review concluded that the global prevalence of duplications of the DMD gene is 11%.[11]

According to these results, the most frequently deleted fragment was exon 45 to exon 52 [Figure 2] followed by exon 45, which was in accordance with the deletion prone 3' hot spot region (major) spanning between exon 44 and 53.[12] In contrast, the proximal deletions were scattered from exon 8 to 32 rather than from exon 2 to 20, which is considered as the 5' hotspot (minor) region. Duplications were confined to the exons 2–13, 3–29, 7–9, 14–27, and exon 31. This pattern was similar to other global populations.[13]

In conclusion, this study for the first time documented the complete spectrum of deletions seen in patients with DMD in a Sri Lankan population and confirms that it corresponds with the pattern seen in other global populations.

Acknowledgements

This project was funded by the NOMA grant of the Human Genetics Unit, Faculty of Medicine, University of Colombo. The authors would like to acknowledge the support of Yasith Mathangasinghe in preparation of this manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

1.
Emery AE. Population frequencies of inherited neuromuscular diseases--a world survey. Neuromuscul Disord 1991;1:19-29.  Back to cited text no. 1
    
2.
Davies KE, Pearson PL, Harper PS, Murray JM, O'Brien T, Sarfarazi M, et al. Linkage analysis of two cloned DNA sequences flanking the Duchenne muscular dystrophy locus on the short arm of the human X chromosome. Nucleic Acids Res 1983;11:2303-12.  Back to cited text no. 2
    
3.
Kingston HM, Sarfarazi M, Thomas NS, Harper PS. Localisation of the Becker muscular dystrophy gene on the short arm of the X chromosome by linkage to cloned DNA sequences. Hum Genet 1984;67:6-17.  Back to cited text no. 3
    
4.
Bushby KM, Thambyayah M, Gardner-Medwin D. Prevalence and incidence of Becker muscular dystrophy. Lancet 1991;337:1022-4.  Back to cited text no. 4
    
5.
Den Dunnen JT, Grootscholten PM, Bakker E, Blonden LA, Ginjaar HB, Wapenaar MC, et al. 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;45:835-47.  Back to cited text no. 5
    
6.
Sellner LN, Taylor GR. MLPA and MAPH: New techniques for detection of gene deletions. Hum Mutat 2004;23:413-9.  Back to cited text no. 6
    
7.
Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: One gene, several proteins, multiple phenotypes. Lancet Neurol 2003;2:731-40.  Back to cited text no. 7
    
8.
Janssen B, Hartmann C, Scholz V, Jauch A, Zshocke J. MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: Potential and pitfalls. J Neurogenet 2005;6:29-35.  Back to cited text no. 8
    
9.
Welihinda J, Karunanayake EH, Jayasekara R, Peiris S, Pettersson U, Wadelius C. Deletion screening of Sri Lankan Duchenne muscular dystrophy patients using the polymerase chain reaction. Ann Trop Paediatr 1993;13:83-6.  Back to cited text no. 9
    
10.
Potnis-Lele DM. Genetic etiology and Diagnostic strategies for Duchenne and Becker Muscular Dystrophy: A 2012 update. Ind J Basic App Med Res 2012;2:357-69.  Back to cited text no. 10
    
11.
Aartsma-Rus A, Ginjaar IB, Bushby K. The importance of genetic diagnosis for Duchenne muscular dystrophy. J Med Genet 2016;53:145-51.  Back to cited text no. 11
    
12.
Oudet C, Hanauer A, Clemens P, Caskey T, Mandel JL. Two hot spots of recombination in the DMD gene correlate with the deletion prone regions. Hum Mol Genet 1992;1:599-603.  Back to cited text no. 12
    
13.
Aartsma-Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJ, Den Dunnen JT. Entries in the Leiden Duchenne muscular dystrophy mutation database: An overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 2006;34:135-44.  Back to cited text no. 13
    


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