Duchenne muscular dystrophy (DMD) is X-linked recessive neuromuscular disorders caused due to mutation in dystrophin gene, leading to progressive muscle weakness. This study was done to identify mutation in dystrophin gene in Nepalese patients with DMD using Multiplex Ligation Dependent Probe Amplification (MLPA) assay in Nepal. Twenty one patients from different regions of Nepal, who were clinically diagnosed as DMD were enrolled in the study. Peripheral blood samples were collected in EDTA vials, gDNA was extracted, and deletion mutation in the dystrophin gene was analysed using Multiplex Ligation Dependent Probe Amplification (MLPA) assay.Exon deletion mutation in the dystrophin gene was observed in 14 (66.6%) out of 21 DMD cases. The most common exon deletion was observed and confined in exon 7-14 and 45-53 of dystrophin gene. The location of deletion in dystrophin gene is apparently non-random with a preponderance found in the hot spot regions. Use of MLPA is useful in detecting copy number changes in DMD proband and suspected carriers in Nepal

Acharya, Bishu

Khatiwada, Saroj

Manandhar, Sulochana

Pokharel, Rohit Kumar

Shrestha, Kushal

Shrestha, Smita

English

Nepal Journal of Biotechnology

Nepal Journal of Biotechnology. Dec .  2016  Vol. 4, No. 1: 37-42   ISSN 2091-1130(Print)/ISSN 2467-9319 (online)  ORIGINAL RESEARCH ARTICLE ©NJB, Biotechnology Society of Nepal    37        Nepjol.info/index.php/njb Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy Kushal Shrestha1, Smita Shrestha2, Mr. Saroj Khatiwada3, Bishnu Acharya4, Sulochana Manandhar5, Rohit Kumar Pokharel6,7 * 1Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal. 2Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal. 3Department of Biochemistry, Modern Technical College, Lalitpur, Nepal. 4Muscular Dystrophy Foundation-Nepal (MDF-Nepal), Lalitpur, Nepal. 5Center for Molecular Dynamics-Nepal (CMDN), Nepal. 6Muscular Dystrophy Foundation-Nepal (MDF-Nepal), Lalitpur. 7Department of Orthopedics and Trauma surgery, IOM, Tribhuvan University, Kathmandu, Nepal. Abstract Duchenne muscular dystrophy (DMD) is X-linked recessive neuromuscular disorders caused due to mutation in dystrophin gene, leading to progressive muscle weakness. This study was done to identify mutation in dystrophin gene in Nepalese patients with DMD using Multiplex Ligation Dependent Probe Amplification (MLPA) assay in Nepal. Twenty one patients from different regions of Nepal, who were clinically diagnosed as DMD were enrolled in the study. Peripheral blood samples were collected in EDTA vials, gDNA was extracted, and deletion mutation in the dystrophin gene was analysed using Multiplex Ligation Dependent Probe Amplification (MLPA) assay. Exon deletion mutation in the dystrophin gene was observed in 14 (66.6%) out of 21 DMD cases. The most common exon deletion was observed and confined in exon 7-14 and 45-53 of dystrophin gene. The location of deletion in dystrophin gene is apparently non-random with a preponderance found in the hot spot regions. Use of MLPA is useful in detecting copy number changes in DMD proband and suspected carriers in Nepal. Keywords: Duchene muscular dystrophy, Multiplex ligation dependent probe amplification, Mutation, Nepal. *Corresponding Author Email: pokharel.rohit@gmail.com  Introduction Duchenne and Becker muscular dystrophies (DMD & BMD) are X-linked recessive neuromuscular disorders with incidence of 1 in 3,500 and 1 in 30,000 live male births, respectively [1]. DMD is characterized by progressive muscle weakness with onset at 3-5 years of age, leading to loss of ambulation by 10-12 years of age without treatment. Respiratory, orthopaedic, and cardiac complications emerge with age, and without intervention, the mean age at death is around 19 years [2]. Both DMD and BMD are caused by mutations in the dystrophin gene (MIM 300377), one of the largest known human genes, spanning about 2.4 Mb of genomic DNA [3]. The most common mutations in the DMD gene are the deletion or duplication of one or more exons [3]. Mutations lead to an absence of or defect in the dystrophin protein, which results in progressive muscle degeneration leading to loss of independent ambulation by the age of 13 years. Variable phenotypic expression relates mainly to the type of mutation and its effect on the production of dystrophin [4]. Muscular dystrophy is diagnosed on the basis of clinical examination, raised creatine phosphokinase (CPK) level, muscle biopsy and dystrophin gene mutation analysis. The genetic tests commonly used to identify dystrophin mutations are multiplex polymerase chain reaction (mPCR), multiplex ligation dependent probe amplification (MLPA), single condition amplification/internal primer, and multiplex amplifiable probe hybridization [5]. Mutation analysis of dystrophin gene in DMD/BMD has been performed in several countries [2, 3]. However, no such studies have been performed in Nepal. MLPA, a simple and cheaper genetic test to detect duplication/ deletion mutation, could be a better tool for genetic diagnosis of DMD in Nepalese patients; thereby assisting in timely diagnosis, treatment and management. We conducted present study among clinically diagnosed DMD patients to find the mutation pattern in dystrophin gene using MLPA and test the efficacy of MLPA in identifying mutation in DMD patients in the Nepalese context. Nepal Journal of Biotechnology. Dec .  2016  Vol. 4, No. 1: 37-42  Shrestha et al.      ©NJB, Biotechnology Society of Nepal    38        Nepjol.info/index.php/njb Materials and Methods  The study was conducted among 21 clinically diagnosed DMD patients from different regions of Nepal, who were referred to Muscular Dystrophy Foundation-Nepal (MDF-Nepal). Diagnosis of DMD was done on the basis of clinical presentations and markedly elevated serum creatine phosphokinase (CPK) levels. Proper genetic counselling was done, and consent was taken from each patient and their parents. Peripheral blood samples (5 ml) were collected from each patient in EDTA vials and transported to laboratory of Central Department of Biotechnology, Tribhuvan University, Kirtipur maintaining cold chain. The samples were stored at -200 C until analysis. Genomic DNA was isolated from blood, as recommended by the MRC-Holland, by using the extraction method of the QIAamp DNA mini kit [6]. The absorbance of DNA samples were measured at 230 nm, 260 nm and 280 nm using the UV spectrophotometer to check for purity of DNA samples required for MLPA assay. A commercial MLPA kit (MRC Holland) with probes of P034 (DMD exons 1-10, 21-30, 41-50, and 61-70) and P035 (DMD exons 11-20, 31-40, 51-60, and 71-79) was used to detect DMD deletion/duplication in DMD patients according to the manufacturer’s recommended protocol (Amsterdam, Netherlands). 50-250 ng of genomic DNA, in a volume of 5 µL Tris-EDTA, was denatured at 980 C for 5 min, cooled down, and then mixed with MLPA P034 or P035 probemix. The mixture was then heated to 950 C for 5 min and incubated at 600 C overnight for probe hybridization. After 16 hours, ligation was performed with Ligase-65 enzyme at 540 C for 15 min and Ligase-65 enzyme was inactivated at 980 C for 5 min. Then, PCR amplification was performed with specific SALSA FAM PCR primers (5’-GGGTTCCCTAAGGGTTGGA-3’). After that, the mixture was separated by capillary electrophoresis and then analyzed using ABI-310 Genetic Analyzer [7]. The Genescan analysis software in the ABI-310 Genetic Analyzer analyzed the raw data to quantify the DNA fragments and determine the size of the fragments by comparing them to fragments in a size standard. The electropherograms of test samples were analyzed by comparing with the peak pattern of the male and female reference samples. The novel software Coffalyser.net was used to analyze the data obtained after the capillary electrophoresis run which was further confirmed by visual assessment by overlaying two fragment profiles and comparing the relative intensities of fragments. The relative peak ratio (RPR) of every single exon was plotted to its corresponding bar chart [8]. Absence of peaks corresponding to two or more contiguous exons was taken to represent a genuine deletion. The absence of only one peak in males, corresponding to a single exon, were also recorded which are yet to be investigated further by applying the novel methods as PCR primer flanking the exons in question or by sequencing. The framedness of the mutation in probands were also estimated by using the dystrophin exonic deletions/duplications reading frame checker 1.9 as recommended by MRC Holland. The data from the study was entered into ms excel and analyzed using SPSS version 11.0. Results  The mean age of DMD patients at the time of study was 11.4 years (age range from 8-18 years). All the patients had very high serum CPK level (median: 6245 U/L, range:  2100-32890 U/L). The genomic DNA extracted from samples had concentration range within 27-59 μg/mL. The ratio of the optical density (OD) of DNA samples at 260 nm and 280 nm ranged from 1.5-2.0 with mean OD 1.76. Among the 21 samples of DMD analyzed by MLPA assay, deletions were observed at various exons of dystrophin gene in 14 samples. Seven samples however did not have any deletion or duplication in the exons. Thus, MLPA tool could detect mutations in about 66.6% (14 of 21 samples). The most prevalent exonic deletion regions were found to be confined in the exon 7-14 and 45-53. Deletion mutations at different exons observed in samples are shown in table 1. Similarly, electropherograms of control sample and test sample after MLPA assay with SALSA probe mix P034 and P035 are shown in figure 1. As indicated by the dystrophin exonic deletions/ duplications reading frame checker 1.9, all the 14 samples with detection of deletion mutation were detected by MLPA assay have out of frame mutations suggesting that the samples were those of DMD patients. But the confirmation of the framedness is yet to be made by gene sequencing, and the 7 samples with negative MLPA result need further genetic testing like sequencing. Nepal Journal of Biotechnology. Dec .  2016  Vol. 4, No. 1: 37-42  Shrestha et al.      ©NJB, Biotechnology Society of Nepal    39        Nepjol.info/index.php/njb  Table 1: Exon deletions detected by MLPA assay in DMD patients Case  Age (years) Serum Creatine kinase level (U/L) Exon deletions Phenotype  1 5 12122 48-50 DMD 2 7 3412 52 DMD 3 6 14467 10-43 DMD 4 8 1287 52 DMD 5 9 11580 45 DMD 6 5 24200 51-53 DMD 7 14 22000 45-50 DMD 8 5 21887 46-49 DMD 9 12 2700 52 DMD 10 11 2100 51 DMD 11 8 4328 12-14 DMD 12 9 3220 51 DMD 13 6 9123 45 DMD 14 7 6245 7-9 DMD  Discussion In Nepal, DMD/BMD patients are generally diagnosed late due to the lack of complete understanding of the disease, poor screening of paediatric group for muscular dystrophy and lack of genetic diagnostic facilities in the country. Most of the cases are diagnosed on the basis of history given by the parents, positive clinical signs of DMD and very high CK level in the blood. For the diagnosis at the gene level clients usually go to or the sample is send outside Nepal, which is not accessible and affordable to many of the parents. This study to detect deletion mutation in DMD using MLPA is first genetic pilot study in Nepal.  Among the 21 samples of clinically diagnosed DMD patients, deletion of dystrophin exon was found in 14 samples (66.6%). However, 7 samples (33.3%) did not show any deletion or duplication. Thus, MLPA was efficient in accurately confirming mutations in about 67% of all the cases. A number of studies have found MLPA assay to be highly sensitive diagnostic assay to identify mutation in DMD and BMD patients [9]. In the study to evaluate the efficacy of MLPA technique in comparison with the traditional multiplex PCR assay in detection of exon deletions and duplications of the DMD gene by Lai et al., MLPA was able to detect all the known deletions and duplications; it detected four additional mutations that had been missed by multiplex PCR  [10]. In a study in China amongst 70 DMD/BMD patients, MLPA detected exonic deletions in 42 patients (60%), exonic duplications in 7 patients (10%) and 21 patients (30%) showed normal results [11]. In the present study, only exons deletion was observed. No exon duplication was found. We found most prevalent exonic deletion regions to be confined in the exons 7-14 and 45-53. One, 3, 4, 6 and 44 exons deletion was observed in 7, 4, 1, 1 and 1 patients respectively. No novel mutations were identified in this study. In a study in Iranian DMD/BMD patients, 30.9 % of patients had single exon deletion, while group and contiguous exon deletions were identified in 41% of the patients [12]. The most numerous exon deletions included exons 45-50, and two exons 3-5 and 41-43 duplications (1.4 %) was observed in a BMD and a DMD patient, respectively [12]. In a study in Polish DMD/BMD patients, Zimowski et al. identified 110 deletions, 22 duplication (in one patient two different duplications were detected) and 2 point mutations. Deletions involved mainly exons 45-54 and 3-21, whereas most duplication involved exons 3-18 [13]. It has been found that deletions account for approximately 60-65% of mutations and duplications for 5-10% in DMD/BMD. The remaining cases are mainly point mutations (30-35% of the cases). Although deletions encompass all 79 exons, two deletion hotspots (exons 45-55 and exons 2-19) are recognized [14]. Identification of exon deletion is important to design gene therapy by exon skipping method that is an emerging therapy for DMD that can transform DMD into BMD is based on the recovery of reading frame induced by alternative splicing of antisense oligonucleotides [15]. More patients may benefit from individual exon skipping therapies following a comprehensive understanding of the correlation between genotypes and phenotypes under the guidance of large-scale genetic epidemiological studies [16]. Similarly identifying disease at earlier stage could help in better management of patients and thus better life quality. This was the first genetic study on DMD in Nepal. The location of deletion in dystrophin gene is apparently non-random with a preponderance found in the hot spot regions in Nepalese population as well. Use of MLPA is useful in detecting copy number changes in DMD proband and suspected carriers.  Nepal Journal of Biotechnology. Dec .  2016  Vol. 4, No. 1: 37-42  Shrestha et al.      ©NJB, Biotechnology Society of Nepal    40        Nepjol.info/index.php/njb  A BA CA Nepal Journal of Biotechnology. Dec .  2016  Vol. 4, No. 1: 37-42  Shrestha et al.      ©NJB, Biotechnology Society of Nepal    41        Nepjol.info/index.php/njb Figure 1. Electropherogram of control sample and test sample after MLPA. Fig. 1A and Fig. 1B are for normal control sample with P034 and P035 respectively. Fig. 1C and Fig. 1D are for DMD patient with deletion in dystrophin gene with P034 and P035 respectively.  Competing Interest None  Acknowledgement We appreciate the parents and DMD boys who gave consent to take part in this study. We acknowledge the support of MDF-Nepal for counseling parents and probands, providing the detail information about the clients and also bearing the cost of some chemicals for gDNA extraction and charge of genetic analyzer. We express our gratitude to Prof. Masafumi Matsuo, M.D; Ph.D. from Kobe, Japan for providing us the MLPA kits, which pioneered the genetic testing of DMD cases in Nepal for the first time. We thank Prof. Tribikram Bhattarai, former head of department of Biotechnology, Tribhuvan University and current head Prof. Dr. Rajani Malla for encouraging and providing the laboratory for the study. We also thank Intrepid Nepal Pvt Ltd.; Centre for Molecular Dynamics (CMDN), Thapathali-11, Kathmandu, Nepal, for allowing us to use the Genetic analyzer at the center. Author’s Contribution KS, SS and RKP designed the study and collected samples. KS, SS, SK, BA and SM engaged in sample collection and conducted laboratory analysis. SK performed statistical analysis and wrote the manuscript. KS, SS and RKP revised the draft. All authors read and approved the final version of the manuscript. References 1. Hegde MR, Chin EL, Mulle JG, Okou DT, Warren ST, Zwick ME: Microarray-based mutation detection in the dystrophin gene. Hum Mutat. 2008, 29(9):1091-99. 2. Li X, Zhao L, Zhou S, Hu C, Shi Y, Shi W, et al: A comprehensive database of Duchenne and Becker muscular dystrophy patients (0-18 years old) in East China. Orphanet J Rare Dis. 2015; 10:5. doi: 10.1186/s13023-014-0220-7. 3. Santos R, Gonçalves A, Oliveira J, Vieira E, Vieira JP, Evangelista T, et al: New variants, challenges and pitfalls in DMD genotyping: implications in diagnosis, prognosis and therapy. J Hum Genet. 2014, 59(8):454-64. 4. Bushby K, Finkel R, Birnkrant DJ, Case LE, Clemens PR, Cripe L, et al: Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management. Lancet Neurol. 2010, 9(1):77-93. 5. Flanigan KM, Dunn DM, von Niederhausern A, Soltanzadeh P, Gappmaier E, Howard MT, et al: Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009, 30(12):1657-66. 6. MLPA, General protocol, MRC-Holland, MLPA, protocol version MDP-v002, last update 23-01-2012. DA Nepal Journal of Biotechnology. Dec .  2016  Vol. 4, No. 1: 37-42  Shrestha et al.      ©NJB, Biotechnology Society of Nepal    42        Nepjol.info/index.php/njb 7. Chen C, Ma H, Zhang F, Chen L, Xing X, Wang S, et al: Screening of Duchenne Muscular Dystrophy (DMD) Mutations and Investigating Its Mutational Mechanism in Chinese Patients. PLoS ONE. 2014, 9(9):e108038. 8. Wang X, Wang Z, Yan M, Huang S, Chen TJ, Zhong N: Similarity of DMD gene deletion and duplication in the Chinese patients compared to global populations. Behav Brain Funct. 2008, 4:20. doi: 10.1186/1744-9081-4-20. 9. Stuppia L, Antonucci I, Palka G, Gatta V: Use of the MLPA Assay in the Molecular Diagnosis of Gene Copy Number Alterations in Human Genetic Diseases. Int J Mol Sci. 2012, 13(3): 3245-76. 10. Lai KKS, Lo IFM, Tong TMF, Cheng LYL, Lam STS: Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA). Clinical Biochemistry. 2006, 9 :367-72. 11. Long F, Sun W, Ji X, Li XH, Liu XQ, Jiang WT, Tao J: Clinical application of multiplex ligation-dependent probe amplification for the detection exonic copy number alterations in the Dystrophin gene. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 201,28(6):699-704. 12. Zamani GR, Karami F, Mehdizadeh M, Movafagh A, Nilipour Y, Zamani M. Analysis of dystrophin gene in Iranian Duchenne and Becker muscular dystrophies patients and identification of a novel mutation. Neurol Sci. 2015. doi:  10.1007/s10072-015-2290-2. 13. Zimowski JG, Massalska D, Holding M, Jadczak S, Fidziańska E, Lusakowska A, et al: MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy. Neurol Neurochir Pol. 2014, 48(6):416-22. 14. Nouri N, Fazel-Najafabadi E, Salehi M, Hosseinzadeh M, Behnam M, Ghazavi MR, et al: Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset. Adv Biomed Res. 2014, 3: 72.  doi: 10.4103/2277-9175.125862. 15. Cirak S, Arechavala-Gomeza V, Guglieri M, Feng L, Torelli S, Anthony K, et al: Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet. 2011, 378(9791):595-605. 16. 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Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy

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Multiplex Ligation Dependent Probe Amplification Based Mutation Analysis of Dystrophin Gene in Nepalese Patients with Duchenne Muscular Dystrophy

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