J. Neurogenetics, 27(1–4): 170–175 Copyright © 2013 Informa Healthcare USA, Inc. ISSN: 0167-7063 print/1563-5260 online DOI: 10.3109/01677063.2013.830616
Exon Deletion Patterns of the Dystrophin Gene in 82 Vietnamese Duchenne/Becker Muscular Dystrophy Patients Van Khanh Tran1, Van Thanh Ta1, Dung Chi Vu2, Suong Thi-Bang Nguyen1, Hai Ngoc Do1, Minh Hieu Ta1, Thinh Huy Tran1 and Masafumi Matsuo3 1 Center for Gene and Protein Research, Hanoi Medical University, Hanoi, Vietnam Department of Medical Genetics, Metabolism and Endocrinology and Clinical Research Division, Research Institute for Child Health Vietnam, National Hospital of Pediatrics, Hanoi, Vietnam 3 Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, Kobe, Japan
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Abstract: Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited muscle diseases caused by mutations in the dystrophin gene. The reading frame rule explains the genotype-phenotype relationship in DMD/BMD. In Vietnam, extensive mutation analysis has never been conducted in DMD/BMD. Here, 152 Vietnamese muscular dystrophy patients were examined for dystrophin exon deletion by amplifying 19 deletion-prone exons and deletion ends were confirmed by dystrophin cDNA analysis if necessary. The result was that 82 (54%) patients were found to have exon deletions, thus confirming exact deletion ends. A further result was that 37 patterns of deletion were classified. Deletions of exons 45–50 and 49–52 were the most common patterns identified, numbering six cases each (7.3%). The reading frame rule explained the genotype-phenotype relationship, but not five (6.1%) DMD cases. Each of five patients had deletions of exons 11–27 in common. The applicability of the therapy producing semifunctional in frame mRNA in DMD by inducing skipping of a single exon was examined. Induction of exon 51 skipping was ranked at top priority, since 16 (27%) patients were predicted to have semifunctional mRNA skipping. Exons 45 and 53 were the next ranked, with 12 (20%) and 11 (18%) patients, respectively. The largest deletion database of the dystrophin gene, established in Vietnamese DMD/BMD patients, disclosed a strong indication for exon-skipping therapy. Keywords: deletion, Duchenne/Becker muscular dystrophy, dystrophin, exon skipping
INTRODUCTION Duchenne (MIM no. 310200) and Becker (MIM no. 300376) muscular dystrophies (DMD/BMD) are frequent genetic diseases, affecting about 1 in every 3500 boys, characterized by progressive muscle wasting. DMD follows severe muscle loss, with patients typically succumbing in their 20s, whereas BMD is an adult-onset mild musclewasting disease. DMD/BMD are caused by mutations in the dystrophin gene, which is the largest gene, spanning ~2.4 Mb of genomic sequence on Xp21.2. The dystrophin gene consists of 79 exons, encoding a 14-kb transcript. It has been reported that deletion and duplication of one or more exons are the most common mutations found in about 60–65% of cases and 5–10% in DMD and BMD, respectively (Takeshima et al., 2010; Lalic et al., 2005; Tran et al., 2005). Deletions cluster in two hotspots: the 5’ terminal and the central regions. Based on these findings, a screening to amplify 19 deletion-prone exons has been
developed (Chamberlain et al., 1988; Beggs et al., 1990). This polymerase chain reaction (PCR)-based screening has been conducted in many countries and deletion pattern differences among races identified (Takeshima et al., 2010; Lalic et al., 2005; Chamberlain et al., 1988; Beggs et al., 1990; White et al., 2002; Lai et al., 2002). The reading frame rule explains the clinical differences between DMD and BMD at the molecular level; the deletions that shift the reading frame of the dystrophin mRNA (out-of-frame) lead to the more severe DMD phenotype, whereas the milder BMD phenotype occurs if the reading frame is preserved (in-frame) (Monaco et al., 1988). This pattern has appeared in ⬎ 90% of cases. However, results obtained by PCR amplification of selected exons did not identify the exact deletion size in all cases. This has made it difficult to examine the reading frame rule in every deletion. To overcome this weak point, dystrophin cDNA examination has been employed as an alternative procedure to disclose exact deletion size
(Received 21 March 2013; accepted 26 July 2013) Address correspondence to Van Khanh Tran, Center for Gene and Protein Research, Hanoi Medical University, 1st Ton That Tung Street, Hanoi 10000, Vietnam. E-mail:
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Dystrophin Deletion in Vietnamese Patients
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(Takeshima et al., 2010). These approaches have enabled investigation of genotype/phenotype correlation. It should be further noted that exact deletion size disclosure in all cases is important for molecular therapy that effectively induces exon skipping to restore the translational reading frame (Takeshima et al., 2006; van Deutekom et al., 2007; Incitti et al., 2010; Aoki et al., 2010). In Vietnam, however, there is no large-scale database for deletions of dystrophinopathy; therefore, strategies for the exon-skipping therapy remain undefined. In this study, we describe the deletion patterns identified in 82 Vietnamese DMD/BMD cases and propose strategies for exon-skipping therapy.
PATIENTS AND METHODS Patients One hundred fifty-two unrelated muscular dystrophy male patients from northern part of Vietnam were referred to the National Institute of Pediatrics in Hanoi from March 2002 to March 2012 for clinical observation and molecular diagnosis. Of the 152 cases, 129 and 23 were clinically diagnosed with DMD and BMD, respectively. Diagnoses were based on typical symptoms, such as pseudohypertrophy, inability to walk independently before the age of 12 or to retain the ability to walk independently after the age of 13, high serum creatine kinase enzyme levels, and known family histories, as described previously (Gilgenkrantz et al., 1989). Informed consent was obtained from all patients’ families for molecular analysis and this study was approved by the ethical committees of Hanoi Medical University (IRB00003121, Hanoi Medical University Institutional Review Board, Hanoi, Vietnam).
Methods Multiplex PCR to amplify 19 deletion-prone exons was used for the first line of mutation detection, as described previously (Chamberlain et al., 1988; Beggs et al., 1990). Amplification was carried out in a total volume of 25 μL containing 100 ng of genomic DNA, 10 mM Tris-HCl (pH 8.3), 50 nM KCl, 5 mM NH4Cl, 250 mM of deoxyribonucleoside triphosphates (Promega, Madison, WI, USA), 1.5 mM MgCl2, 6.25 pmol of each primer, and 2 units of Taq polymerase (PerkinElmer Cetus, Norwalk, CT, USA). PCR cycling conditions were as follows: an initial denaturation at 95°C for 7 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55–62°C for 50 s, and extension at 72°C for 50 s, and a final extension at 72°C for 5 min. The PCR products were resolved by electrophoresis in a 2% agarose gel containing 0.5 μg/mL ethidium bromide. All samples were analyzed in two separate
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reactions. Positive and negative controls were included using a normal genomic DNA sample and blank buffer, respectively. Dystrophin cDNA was subjected for analysis to identify deletion and end points. For this purpose, RNA was extracted from fresh blood and reverse transcription PCR was used to analyze dystrophin expressed in lymphocyte, as mentioned previously (Matsuo et al., 1991; Roberts et al., 1991). Reverse transcriptase (RT)-PCR amplification and direct sequencing were performed to define deletion boundaries. The primers used for RT-PCR and sequencing analysis were described previously (Nishiyama et al., 2008). PCR products were directly sequenced after gel purification (Novagen, Madison, WI, USA) (Tran et al., 2006, 2007). The DNA sequence was determined using an automated DNA sequencer (model 373A; Applied Biosystems, Foster City, CA, USA). When mutations were identified in cDNA, they were confirmed for deletion breakpoints in genomic DNA by PCR amplification of the corresponding genomic region with the primers mentioned previously (Bennett et al., 2001).
RESULTS In this study, 82 male patients with deletion in the dystrophin gene were examined for clinical features and creatine kinase (CK) levels (Table 1). The Gower’s sign were positive in all of the patients. About 84% of the patients showed pseudohypertrophy of the calf. Serum creatine kinase (CK) levels were elevated in all cases, with minimum level at 2600 U/L. Deletion screening by amplifying 19 deletion-prone exons of the dystrophin gene disclosed nonamplification in one or more exons in 82 cases (54%) among 152 unrelated patients. The exact deletion end points were disclosed in 27 patients but not in 55 patients in whom dystrophin cDNA covering the nonamplified exon was examined. Therefore, exact deletion ends were determined in all exon deletions. The resulting deletions were classified into 37 patterns; 34 patterns in 75 DMD and 3 patterns in 7 BMD (Figure 1A–C). Among the 37 deletion patterns, the most frequently appearing was the out-of-frame deletion of exons 45–50 and 49–52, each were found in 6 cases (7.3%); all being DMD (Figure 1A). The next most common patterns were in-frame deletion of exons 45–47 found in 5 BMD cases and out-of-frame deletions of exons 48–50 or 49–50 found in 5 DMD cases each (6.1%) (Figure 1D). The largest deletion encompassed exons 3–44 found in 1 case. A single-exon deletion was found in 4 patterns among 14 cases (17.1%). Exon 51 deletion was the most common single-exon deletion and was found in 5 cases (6.1%), all being DMD. The second most common single-exon deletion removed exon 44, identified in 4 DMD cases (4.9%) (Figure 1D). Deletion starting in the central hotspot region (exons 40–60) represented 72% (59/82) of identified
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Table 1. Genotype and phenotype of Vietnamese DMD/BMD patients in this study.
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Genotype Mutation
n
Type
Current Age (year)
Del 2–13 Del 3–7 Del 3–34 Del 3–44 Del 5–27 Del 5–37 Del 7–12 Del 8–9
2 3 1 1 1 1 2 1
out out in in in in out out
9, 11 11, 13, 15 10 dead dead 11 10, 12 dead
5 5–6 4 5 6 5 7, 8 7
Del 8–12 Del 8–44 Del 10–17 Del 10–43 Del 11–39 Del 18–47 Del 32–43 Del 33–43 Del 38–43 Del 42–43 Del 43–52 Del 44
1 1 1 1 1 1 1 1 1 2 1 4
out out out out in out out out out out out out
10 13 12 17 dead 11 7 15 11 7, 8 7 8, 10, 12, 13
6 6 5 5 7 6 6 7 7 6, 7 7 5–7
Del 45–52 Del 45 Del 45–50
2 2 6
out out out
2, 5 7, 8 6–7
Del 46–47 Del 46–50
3 3
out out
10, 12 9, 11 9, 11, 12, 13, 14, 14 10, 13, 15 9, 11, 13
Del 46–51 Del 46–55 Del 48–50 Del 49–50
4 1 5 5
out out out out
6, 10, 10, 12 11 8, 8, 8, 10, 12 6, 8, 8, 12, 13
6–8 7 4–7 3–7
Del 49–52
6
out
9, 10, 10, 11, 12, 12
5–7
Del 50–52 Del 51
1 5
out out
12 2, 6, 9, 8, 12
7 2–7
Del 51–53 Del 52 Del 3–8 Del 45–48 Del 45–47
1 3 1 1 5
out out in in in
13 7, 8, dead 12 22 11, 14, 16, 20, 29
8 5–7 8 8 8–10
Clinical information
Onset (year)
CK U/l ⬎ 10000 ⬎ 9000 13000 29130 90127 13000 ⬎ 10000 14230
7–8
Best current motor ability
Frequent falling, walking on toe Walking on toe. wheelchair bound Difficulty climbing stair Bed-ridden, walking on toe Wheelchair bound Frequent falling, walking on toe Frequent falling, walking on toe Not able to run or can not climb stair 11345 Waddling gait 20150 Wheelchair bound 12177 Waddling gait, can not climb stair 9800 Wheelchair bound 12124 Wheelchair bound 11700 Frequent falling, wheelchair bound 11000 Frequent falling 2600 Wheelchair bound 7500 Frequent falling, walking on toe ⬎ 10000 Frequent falling, waddling gait 9520 Walking on toe ⬎ 10000 Frequent falling, walking on toe. wheelchair bound ⬎ 10000 Frequent falling, walking on toe ⬎ 9500 waddling gait ⬎ 7000 Frequent falling, walking on toe wheelchair bound ⬎ 9500 Frequent falling, wheelchair bound ⬎ 7000 Waddling gait, wheelchair bound ⬎ 9000 12500 ⬎ 10000 ⬎ 9500
Walking on toe. wheelchair bound Frequent falling Walking on toe Walking on toe. waddling gait, wheelchair bound ⬎ 7500 Frequent falling, walking on toe. waddling gait 13540 Frequent falling, walking on toe ⬎ 10000 Cannot sit at 10 months, walking on toe, frequent falling, wheelchair bound 11000 Walking on toe, wheelchair bound ⬎ 9500 Frequent falling 3500 Still able to walk 5780 Still able to walk, waddling gait ⬍ 6000 Still able to walk, waddling gait
Lower limb muscle
Phenotype
Calf pseudohypertrophy Calf pseudohypertrophy Calf pseudohypertrophy Progressive muscle weakness Calf pseudohypertrophy Calf pseudohypertrophy Progressive muscle weakness Calf pseudohypertrophy
DMD DMD DMD DMD DMD DMD DMD DMD
Calf pseudohypertrophy Calf pseudohypertrophy Calf pseudohypertrophy Muscle wasting Muscle wasting Calf pseudohypertrophy Calf pseudohypertrophy Muscle wasting Calf pseudohypertrophy Progressive muscle weakness Calf pseudohypertrophy Calf pseudohypertrophy
DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD
Calf pseudohypertrophy Calf hypertrophy Contracture reflexes depressed, calf pseudohypertrophy Contracture reflexes depressed Progressive muscle weakness, calf hypertrophy Calf pseudohypertrophy Contracture reflexes depressed Calf pseudohypertrophy Calf pseudohypertrophy, muscle wasting Calf pseudohypertrophy
DMD DMD DMD
Calf pseudohypertrophy Calf pseudohypertrophy
DMD DMD
Muscle wasting Calf pseudohypertrophy Calf pseudohypertrophy Calf pseudohypertrophy Calf pseudohypertrophy
DMD DMD BMD BMD BMD
DMD DMD DMD DMD DMD DMD DMD
Note. n ⫽ number of patients; out ⫽ out-of-frame; in ⫽ in-frame; CK ⫽ creatinine kinase level. Grower’s sign was positive in all the cases.
deletions, whereas deletion starting in the proximal hotspot (exons 2–20) accounted for only 13.4% (11/82). Gross deletions spanning the proximal to central regions were identified in 12 cases (14.6%) The genotype/phenotype correlation was examined by determining their reading frame. The in-frame deletion was identified in all 7 BMD cases. However, the in-frame deletion was also identified in 5 DMD cases (Figure 1B and C). These 5 deletions did not conform to the reading
frame theory. Although 70 DMD cases had out-of-frame deletions conforming to the reading frame theory, 5 of the 82 total population (6.1%) did not conform. It was remarkable that all 5 had deletion of exons 11–27 in common. Currently, induction of exon 51 skipping using antisense oligonucleotide is now on a phase II clinical trial (Cirak et al., 2011; Kinali et al., 2009). Therefore, we examined applicability of exon-skipping therapy in Vietnamese DMD patients. Exon 51 skipping was found applicable in
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Figure 1. Patterns of exon deletion in Vietnamese DMD and BMD cases. (A) Structural domains of the human full-length dystrophin. Exon deletions were categorized into 37 patterns: 3 and 34 patterns were identified in (B) BMD and (C) DMD, respectively. Deleted exon regions are represented by horizontal bars. Black or gray bars represent out-of-frame or in-frame deletions, respectively. On the bottom line, exons are numbered 1–79. The number at the end of each bar represents the number of cases with an identical deletion. (D) Distribution of major deletion patterns in DMD/BMD patients. Numbers indicated the percentage of patterns.
16 cases with 3 deletion patterns (exons 45–50 deletion in 6 cases, exons 48–50 deletion in 5 cases, and exons 49–50 deletion in 5 cases) (Table 2). Induction of exon 45 and exon 53 skipping were found applicable to 12 and 11 patients, respectively. Induction of exon 50, 44, 43, or 8 then was nominated as a potential target of exon-skipping therapy.
DISCUSSION In the present study, the screening of deletion-prone exon of dystrophin gene by PCR application of 19 Table 2. Applicable cases for exon skipping therapy. Molecular therapies Skipping of Exon 51 Exon 45 Exon 53 Exon 50 Exon 44 Exon 43 Exon 8
Applicable cases
16 12 11 6 4 4 3
(27%) (20%) (18%) (10%) (7%) (7%) (5%)
deletion-prone exons successfully disclosed deletion in 82 (54%) Vietnamese DMD/BMD cases. This largest deletion database of the dystrophin gene in Vietnamese DMD/BMD patients was established for the first time. This is very important to provide correct diagnosis or genetic counseling for all our dystrophinopathy cases and will also expedite the application of specific exonskipping gene therapies. It has been reported that about one third of deletions were clustered at the proximal hotspot, with the remainder located at the central hotspot (http://www.dmd.nl/DMD_ home.html). Our study does not differ from populations reported in the literature, although the deletion frequency appeared to be slightly lower in Vietnamese patients. This may come from inclusion of the other type of muscle disease because the criteria for selection of the patients were totally clinically based. In Vietnam, the most frequent single-exon deletions were exon 51 (6.1%) and exon 44 (4.9%). The rates of single-exon deletion were 17.1% in Vietnam, compared with 15% in Japan and about 15–20% in represented Caucasian populations (Okizuka et al., 2009; Takeshima et al., 2010; Carsana et al., 2005; Gilgenkrantz et al., 1989; Schwartz et al., 2004). In the Leiden database
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summary, involvement of intron 44 was found to be frequently involved in the development of the deletion (http://www.dmd.nl/DMD_home.html). Accurate identification of the deletion end point is important not only for examining genotype/phenotype correlation but also for establishing strategies for molecular therapy. In the present study, we successfully identified the deletion end point in all cases by combining dystrophin cDNA amplification and sequencing analysis. These deletion breakpoints were further confirmed in genomic DNA analysis of the corresponding genomic region to avoid exon skipping in the ordinary splicing. Although the reading frame rule is thought to explain the phenotype (Monaco et al., 1988; Koenig et al., 1989), 5 out of 82 cases with deletion (6.1%) did not fit with the reading frame rule (Figure 1). Since all deletions removed exons 11–27 in common, this common region may work to exempt the rule. Induction of exon skipping with antisense oligonucleotide is a promising method for restoration of dystrophin expression in DMD therapy. Skipping of exon 51 is now undergoing clinical trials (Cirak et al., 2011; Kinali et al., 2009). In Vietnam, it was found that skipping of exon 51 is the first priority for DMD therapy. Our data fully illustrate therapeutic strategies that would be considered for the treatment of the Vietnamese Duchenne muscular dystrophy in the near future.
ACKNOWLEDGMENTS We thank Drs. V. Fitzmaurice and T. T. Vu for critically reading of the manuscript; And the patients and their families for their voluntary involvement in this study. This work was supported by National Foundation for Science and Technology Development (NAFOSTED) research fund, Vietnam. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES Aoki, Y., Nakamura, A., Yokota, T., Saito, T., Okazawa, H., Nagata, T., & Takeda, S. (2010). In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52–deficient mdx mouse. Mol Ther, 18, 1995–2005. Beggs, A. H., Koenig, M., Boyce, F. M., & Kunkel, L. M. (1990). Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet, 86, 45–48. Bennett R. R., den Dunnen J., O’Brien, K. F., Darras, B. T., & Kunkel, L. M. (2001). Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing. BMC Genet, 2, 17.
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