The spectrum of deletions and duplications in the dystrophin (DMD) gene in a cohort of patients with Duchenne muscular dystrophy in Sri Lanka
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.263235
Source of Support: None, Conflict of Interest: None
Keywords: Deletions, Duchenne muscular dystrophy, duplications, multiple ligation binding probe amplification, Sri Lanka
Duchenne muscular dystrophy (DMD) is a rapidly progressive X-linked recessive neuromuscular disorder with a worldwide incidence of 1/3500 among male live births. 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). 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., 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. Testing for mutations in large genes, such as in the DMD gene, has been a challenging venture until recently. 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. 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. 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.
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.
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].
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].
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].
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. 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. According to a systematic review conducted in 2016, the prevalence of mutations of the DMD gene is approximately 68%. 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%.
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. 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.
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.
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
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2]