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Molecular analysis of the dystrophin gene in 407 Chinese patients with Duchenne/Becker muscular dystrophy by the combination of multiplex ligation-dependent probe amplication and Sanger sequencing , ☆☆ Wan-Jin Chen a, b, , Qi-Fang Lin a , Qi-Jie Zhang a , Jin He a , Xin-Yi Liu a , Min-Ting Lin a , Shen-Xing Murong a , Chia-Wei Liou c , Ning Wang a, b a Department of Neurology and Institute of Neurology, First Afliated Hospital, Fujian Medical University, Fuzhou, Fujian, China b Center of Neuroscience, Fujian Medical University Fuzhou, Fujian, China c Department of Neurology, Mitochondrial Research Unit, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan abstract article info Article history: Received 21 November 2012 Received in revised form 5 April 2013 Accepted 6 April 2013 Available online 13 April 2013 Keywords: Duchenne/Becker muscular dystrophy Dystrophin gene Genetic diagnosis Genotypephenotype Multiplex ligation-dependent probe amplication Sanger sequence Background: Progressive muscular dystrophy is a leading neuromuscular disorder without any effective treat- ments and a common genetic cause of mortality among teenagers. A challenge exists in the screening of subtle mutations in 79 exons and little is known about the genotype-phenotype correlation. Methods: Here we adopted multiplex ligation-dependent probe amplication and Sanger sequencing to detect the dystrophin gene in 407 patients and 76 mothers. Results: Sixty-three percent (257/407) of the patients harbored a deletion or duplication mutation, with a de novo mutation frequency of 39.5% in 76 affected patients, and approximately 43.7% of the deletions occurred from exon 45 to 52. To those patients suspected with single exon deletion, combined with Sanger sequencing, ve subtle mutations were identied: c.8608C > T, c.2302C > T, c.7148dupT, c.10855C > T and c.2071-2093del AGGGAACAGATCCTGGTAAAGCA; the last three mutations were novel. Furthermore, after genotypephenotype analysis, the severity of DMD/BMD was associated with the frame shift mutation but not with the deletion, the duplication or the number of deleted exons. Conclusion: The majority of patients have a deletion/duplication mutation in the dystrophin gene, with a hot de- letion mutation region from exon 45 to 52. Combined with Sanger sequencing, multiplex ligation-dependent probe amplication is capable of detecting part of subtle mutations. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Duchenne/Becker muscular dystrophy (DMD/BMD) is the most com- mon X-linked recessive inherited neuromuscular disease, with a high fre- quency of 1 in 3500 boys [1]. The severe form, DMD is characterised by progressive muscle weakness, pseudo-hypertrophy in calf muscle and Gowers' sign [2]; patients are usually diagnosed at the age of 5 and die of respiratory failure or cardiomyopathy near 20 years of age [3]. With a milder manifestation and slow progression, BMD patients are capable of walking independently at 12 years of age and have a normal life expec- tancy [4]. The disease-causing gene, dystrophin, which is located in Xq21 with 79 exons, encodes a membrane protein called dystrophin [5]. Mutations in this gene are responsible for the differences between DMD and BMD. When a frame shift mutation occurs and produces a non-functional protein, the patients suffer from DMD; in contrast, when the reading frame is maintained and encodes a partially functional protein, the patients will show the milder clinical symptoms of BMD [6]. Based on this reading-frame rule, restoration of the open reading frame (ORF) using a morpholino oligomer is a major strategy to treat DMD patients now [7]. Although multiplex PCR has been widely used in genetic diagnosis of DMD/BMD, it is time-consuming and difcult to cover all of the exons. In addition, approximately 10% patients with duplication muta- tions will be misdiagnosed [8]. In 2002, multiplex ligation-dependent probe amplication (MLPA), was invented by Schouten [9], which possesses the capacity to quantify all 79 exons in only 2 reactions and facilitates the diagnosis of DMD/BMD. Interestingly, for certain subtle mutations at the probe ligation site, failure probe hybridisation Clinica Chimica Acta 423 (2013) 3538 Author contributions: Study concept and design (Drs. W-J Chen and Wang); ac- quisition of data (Drs. W-J Chen, Q-F Lin, Zhang, He, Liu, M-T Lin, Murong, Liou and Wang); analysis and interpretation of data (Drs. W-J Chen, Q-F Lin, Zhang, He, Liu and Wang); drafting of the manuscript (Drs.W-J Chen, Q-F Lin and Zhang); obtaining of funding (Drs. W-J Chen and Wang); administrative, technical, or material support (Drs. W-J Chen, Q-F Lin, Zhang, He, Liu and Wang); study supervision (Dr. Chen). ☆☆ Disclosure: The authors report no conicts of interest. Corresponding author at: Department of Neurology, First Afliated Hospital, Fujian Medical University, 20 Chazhong Road, Fuzhou 350005, China. Tel.: +86 591 87982772; fax: +86 591 83375472. E-mail address: [email protected] (W.-J. Chen). 0009-8981/$ see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.04.006 Contents lists available at SciVerse ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
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Page 1: Clinica Chimica Acta - download.xuebalib.comdownload.xuebalib.com/xuebalib.com.50444.pdf · can occur and lead to confusion with the deletion mutation, which enables MLPA to detect

Clinica Chimica Acta 423 (2013) 35–38

Contents lists available at SciVerse ScienceDirect

Clinica Chimica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /c l inch im

Molecular analysis of the dystrophin gene in 407 Chinese patients withDuchenne/Becker muscular dystrophy by the combination of multiplexligation-dependent probe amplification and Sanger sequencing☆,☆☆

Wan-Jin Chen a,b,⁎, Qi-Fang Lin a, Qi-Jie Zhang a, Jin He a, Xin-Yi Liu a, Min-Ting Lin a, Shen-Xing Murong a,Chia-Wei Liou c, Ning Wang a,b

a Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, Chinab Center of Neuroscience, Fujian Medical University Fuzhou, Fujian, Chinac Department of Neurology, Mitochondrial Research Unit, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan

☆ Author contributions: Study concept and design (quisition of data (Drs. W-J Chen, Q-F Lin, Zhang, He, LWang); analysis and interpretation of data (Drs. W-Jand Wang); drafting of the manuscript (Drs.W-J Chen,of funding (Drs. W-J Chen and Wang); administrative,(Drs. W-J Chen, Q-F Lin, Zhang, He, Liu and Wang); stud☆☆ Disclosure: The authors report no conflicts of inter

⁎ Corresponding author at: Department of NeurologyMedical University, 20 Chazhong Road, Fuzhou 350005, Cfax: +86 591 83375472.

E-mail address: [email protected] (W.-

0009-8981/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.cca.2013.04.006

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 21 November 2012Received in revised form 5 April 2013Accepted 6 April 2013Available online 13 April 2013

Keywords:Duchenne/Becker muscular dystrophyDystrophin geneGenetic diagnosisGenotypephenotypeMultiplex ligation-dependent probeamplificationSanger sequence

Background: Progressive muscular dystrophy is a leading neuromuscular disorder without any effective treat-ments and a common genetic cause of mortality among teenagers. A challenge exists in the screening of subtlemutations in 79 exons and little is known about the genotype-phenotype correlation.Methods: Here we adopted multiplex ligation-dependent probe amplification and Sanger sequencing to detectthe dystrophin gene in 407 patients and 76 mothers.Results: Sixty-three percent (257/407) of the patients harbored a deletion or duplication mutation, with a denovo mutation frequency of 39.5% in 76 affected patients, and approximately 43.7% of the deletions occurredfrom exon 45 to 52. To those patients suspected with single exon deletion, combined with Sanger sequencing,five subtlemutationswere identified: c.8608C > T, c.2302C > T, c.7148dupT, c.10855C > T and c.2071-2093delAGGGAACAGATCCTGGTAAAGCA; the last three mutations were novel. Furthermore, after genotype–phenotypeanalysis, the severity of DMD/BMD was associated with the frame shift mutation but not with the deletion, theduplication or the number of deleted exons.Conclusion: The majority of patients have a deletion/duplication mutation in the dystrophin gene, with a hot de-

letion mutation region from exon 45 to 52. Combined with Sanger sequencing, multiplex ligation-dependentprobe amplification is capable of detecting part of subtle mutations.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Duchenne/Becker muscular dystrophy (DMD/BMD) is the most com-mon X-linked recessive inherited neuromuscular disease, with a high fre-quency of 1 in 3500 boys [1]. The severe form, DMD is characterised byprogressive muscle weakness, pseudo-hypertrophy in calf muscle andGowers' sign [2]; patients are usually diagnosed at the age of 5 and dieof respiratory failure or cardiomyopathy near 20 years of age [3]. With amilder manifestation and slow progression, BMD patients are capable of

Drs. W-J Chen and Wang); ac-iu, M-T Lin, Murong, Liou andChen, Q-F Lin, Zhang, He, LiuQ-F Lin and Zhang); obtainingtechnical, or material supporty supervision (Dr. Chen).est., First Affiliated Hospital, Fujianhina. Tel.: +86 591 87982772;

J. Chen).

rights reserved.

walking independently at 12 years of age and have a normal life expec-tancy [4].

The disease-causing gene, dystrophin, which is located in Xq21 with79 exons, encodes amembrane protein called dystrophin [5]. Mutationsin this gene are responsible for the differences between DMD and BMD.When a frame shift mutation occurs and produces a non-functionalprotein, the patients suffer from DMD; in contrast, when the readingframe is maintained and encodes a partially functional protein, thepatients will show the milder clinical symptoms of BMD [6]. Based onthis reading-frame rule, restoration of the open reading frame (ORF)using a morpholino oligomer is a major strategy to treat DMD patientsnow [7].

Although multiplex PCR has been widely used in genetic diagnosisof DMD/BMD, it is time-consuming and difficult to cover all of theexons. In addition, approximately 10% patients with duplication muta-tions will be misdiagnosed [8]. In 2002, multiplex ligation-dependentprobe amplification (MLPA), was invented by Schouten [9], whichpossesses the capacity to quantify all 79 exons in only 2 reactionsand facilitates the diagnosis of DMD/BMD. Interestingly, for certainsubtle mutations at the probe ligation site, failure probe hybridisation

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36 W.-J. Chen et al. / Clinica Chimica Acta 423 (2013) 35–38

can occur and lead to confusion with the deletion mutation, whichenables MLPA to detect certain subtle mutations. Here, we screen thedystrophin gene mutations in a large cohort of 407 DMD/BMD patientsin China using MLPA to explore the mutation distribution and identifya reliable genotype-phenotype correlation.

2. Materials and methods

2.1. Samples

From June 1997 to June 2012, 407 patients from 166 unrelatedDMD/BMD Chinese Han families were recruited. All of the patientsfulfilled the DMD/BMD diagnosis criterion [10] andwere further dividedinto 3 types based on their age of wheelchair dependency: DMD (before12 years), IMD (13–15 years) and BMD(after 16 years) [11]. 76motherswere enrolled for carrier screening. In addition, 20 healthy males and 20healthy females were selected as controls for theMLPA analysis, and an-other 100 healthy controls (50 males and 50 females) were selected toconfirm the three novel mutations that we reported in this manuscript.

Fig. 1. The five subtle mutations from five unrelated families confirmed by Sanger seqAGGGAACAGATCCTGGTAAAGCA mutation in exon 17 (c, patient; d, mother; e, father) c.819 (h, patient; I, mother) c.10855C > T mutation in exon 76 (j, patient).

Written informed consent was obtained from every participant or theirguardian, and our study was approved by the ethics committee of theFirst Affiliated Hospital of Fujian Medical University.

2.2. MLPA analysis

MLPA was carried out using the SALSA MLPA kit P034/P035 DMD(MRC-Holland®). After denaturation, hybridisation, ligation and ampli-fication, the products were separated using capillary electrophoresis(ABI 3130). The raw data were analysed using Genemapper 3.0 andExcel software, and the copy number was calculated according to theMLPA kit instruction (http://www.mlpa.com).

2.3. PCR and Sanger sequencing

For the samples with a suspected single exon deletion, PCR and di-rect sequencing were employed to clarify the occurrence of subtle mu-tations at the probe ligation site. If the result of PCR shows no deletion,

uencing. c.7148dupT mutation in exon 49 (a, patient; b, mother) c.2071-2093del608C > T mutation in exon 58 (f, patient; g, mother) c.2302C > T mutation in exon

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Table 1Correlation between dystrophin mutation types and disease severity.

Mutation types Disease severity Total χ2/P

DMD IMD BMD

Frame shift mutation 148 26 8 182 122.53 P = 0.000Non-frame shift mutation 16 12 52 80Total 164 38 60 262Deletion mutation 136 29 52 217 1.299 P = 0.522Duplication mutation 24 8 8 40Total 160 37 60 257One exon deletion 21 9 4 34 1.692 P = 0.429Two–three exons deletion 34 5 20 59Four–five exons deletion 29 5 15 49Six–seven exons deletion 19 3 6 28Eight exons deletion 33 7 17 47Total 136 29 52 217

37W.-J. Chen et al. / Clinica Chimica Acta 423 (2013) 35–38

it's reasonable to suspect the sample carry a subtle mutation, and canfurther be confirmed by Sanger sequencing.

2.4. Data statistics

The genotype–phenotype correlation was analysed by Chi-squaretest using SPSS 16.0. P value b0.05 (two-tailed) was considered tobe statistically significant.

3. Results

3.1. Mutations of dystrophin gene

Among the 407 clinically suspected DMD/BMD patients, 217 onespossessed one to eight exons deletions and 40 had duplications. Com-bined with PCR and Sanger sequencing, 5 patients with a copy numberratio below 0.7 carried 5 subtle mutations: c.8608C > T, c.7148dupT,c.2302C > T, c.10855C > T, c.2071-2093del AGGGAACAGATCCTGGTAAAGCA (Fig. 1); the latter three mutations were novel and notdetected in 150 X chromosomes from 100 healthy controls. However,in the other 145 patients, neither deletion nor duplication mutationswere detected. To identify the hot mutation regions in dystrophin, wecalculated the deletion and duplication frequency for each exon. Thiscalculation revealed that most of the deletions (43.7%) occurred inexons 45 to 52 (Fig. 2a), whereas no frequent duplication exons werefound (Fig. 2b).

3.2. Results of carrier status

Among the 76 mothers, 63 were the mothers of patients with exondeletions and the rest 13 were those with exon duplications. AfterMLPA analysis, 46 mothers possessed the same deletion (36/63) orduplication (10/13) mutations as their children; the remaining 30mothers were normal, indicating that the de novomutation frequencywas 39.5% (30/76).

3.3. Analysis of the genotype–phenotype association

All of the patients were divided into DMD, IMD and BMD to furtherinvestigate the correlation between disease severity and mutation typeand extent of deletion. The results showed that ORF lostwas a highly re-lated factor to the disease severity (χ2 = 122.53, P b 0.001; Table 1).

Fig. 2. The frequency of deletion and duplication mutation for all 79 exons in dystrophingene. The deletion mutation can occur in every exon especially in a mutation rich regionfrom exon 45 to 52 (2a). As to duplication mutation, from exon 2 to 63 can occur withoutsignificant frequent region, but none harbors duplication mutation in exon 1, 64 to 79 inthis cohort of patients (2b).

However, no significant differences were found in the distributions ofdeletion and duplication mutation among different types of patients(χ2 = 1.299, P = 0.522; Table 1). The correlation of disease severityand the extent of deletion was not statistically remarkable as well(χ2 = 1.692, P = 0.429; Table 1).

4. Discussion

After mutation analysis of the dystrophin gene in 407 DMD/BMDchildren and 76 mothers, 257 of the affected patients possesseddeletion/duplication mutations, with a de novo mutation frequencyof 39.5% and a mutation-rich area (exon 45–52). We further investi-gated the relationship between phenotype severity and mutationtypes. Among the 262 genetically confirmed patients, the frameshift mutations were highly associated with the disease severity. Itis clear that a frame shift mutation tends to profoundly change theamino acid sequence and encode a non-functional protein. Approxi-mately 90.2% (148/164) of DMD patients and 86.7% (52/60) of BMDpatients followed this reading-frame rule, which was consistent witha previous study [11], the remaining 24 patients may be attributed toalternative splicing, exon skipping or an internal promoter [12,13].Except for frame shift mutations, deletions/duplications and the num-ber of deleted exons did not seem to influence the progression of thedisease, which is similar to the findings of Magri et al. [14].

Challenges remain in the detection of subtle mutations among the79 exons, which are often misdiagnosed using multiple PCR or MLPA.Because they are time-consuming and cost-prohibitive, few studieson subtle mutations have been performed. In our study, the patientswith a ratio below 0.7 were further screened for point mutationsusing PCR and Sanger sequencing, and five subtle mutations wereconfirmed. Among these mutations, c.8608C > T, c.2302C > T, andc.10855C > T give rise to a premature stop codon, whereas c.7148dupTand c.2071-2093del AGGGAACAGATCCTGGTAAAGCA produce frameshift mutations. However, for the other 145 patients who did not exhibitexon deletions or duplications, subtle mutation screening is a hard pro-cedure if it depends on direct Sanger sequencing. Fortunately, usingnext-generation sequencing, Xie S et al. detected a c.10141C > T muta-tion in a Chinese DMD pedigree [15], which offers a new promising ave-nue for the efficient detection of subtle mutations.

In conclusion, MLPA is a fast, accurate and reliable deletion/duplication mutation screening method for the dystrophin gene.ORF disruption is ameaningful prognostic factor for progressivemusculardystrophy. For the patients with a suspected single exon deletion, MLPAcombined with Sanger sequencing offers the capability to distinguish aportion of the subtle mutations at the probe ligation site. Although con-siderable progress has been made in the diagnosis of DMD/BMD usingMLPA, the detection of subtle mutations among the 79 exons remains acumbersome procedure.

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38 W.-J. Chen et al. / Clinica Chimica Acta 423 (2013) 35–38

Acknowledgments

The authors sincerely thank the DMD/BMD families for their helpand willingness to participate in this study. This work was supportedby the grant 2012 J06016 from the Natural Science Foundation ofFujian Province of China, grant JA12129 from the Program for NewCentury Excellent Talents in Fujian Province University, a Program forclinical medical key discipline of Fujian Medical University (XK201108),a key program of scientific research of Fujian Medical University(2009D064), and a key clinical specialty discipline construction pro-gram of Fujian.

References

[1] Manzur AY, Muntoni F. Diagnosis and new treatments in muscular dystrophies.Postgrad Med J 2009;85:622–30.

[2] Bushby K, Finkel R, Birnkrant DJ, et al. Diagnosis and management of Duchennemuscular dystrophy, part 1: diagnosis, and pharmacological and psychosocialmanagement. Lancet Neurol 2010;9:77–93.

[3] Essex C, Roper H. Lesson of the week: late diagnosis of Duchenne's muscular dys-trophy presenting as global developmental delay. BMJ 2001;323:37–8.

[4] Bushby KM, Gardner-Medwin D, Nicholson LV, et al. The clinical, genetic and dys-trophin characteristics of Becker muscular dystrophy. II. Correlation of phenotypewith genetic and protein abnormalities. J Neurol 1993;240:105–12.

[5] Roberts RG. Dystrophin, its gene, and the dystrophinopathies. Adv Genet 1995;33:177–231.

[6] Koenig M, Beggs AH, Moyer M, et al. The molecular basis for Duchenne versusBecker muscular dystrophy: correlation of severity with type of deletion. Am JHum Genet 1989;45:498–506.

[7] Kinali M, Arechavala-Gomeza V, Feng L, et al. Local restoration of dystrophin expres-sion with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: asingle-blind, placebo-controlled, dose-escalation, proof-of-concept study. LancetNeurol 2009;8:918–28.

[8] Hallwirth Pillay KD, Bill PL, Madurai S, et al. Molecular deletion patterns inDuchenne and Becker muscular dystrophy patients from KwaZulu Natal. J NeurolSci 2007;252:1–3.

[9] Schouten JP, McElgunn CJ, Waaijer R, et al. Relative quantification of 40 nucleicacid sequences by multiplex ligation-dependent probe amplification. Nucleic AcidsRes 2002;30:e57.

[10] Emery AEH. Duchenne muscular dystrophy.New York: Oxford University Press;1993392–8 [New York].

[11] Tuffery-Giraud S, Béroud C, Leturcq F, et al. Genotype-phenotype analysis in 2,405patients with a dystrophinopathy using the UMD-DMD database: a model of na-tionwide knowledgebase. Hum Mutat 2009;30:934–45.

[12] Gualandi F, Rimessi P, Trabanelli C, et al. Intronic breakpoint definition and transcrip-tion analysis in DMD/BMD patients with deletion/duplication at the 5’mutation hotspot of the dystrophin gene. Gene 2006;370:26–33.

[13] Kesari A, Pirra LN, Bremadesam L, et al. Integrated DNA, cDNA, and protein studiesin Becker muscular dystrophy show high exception to the reading frame rule.Hum Mutat 2008;29:728–37.

[14] Magri F, Govoni A, D'Angelo MG, et al. Genotype and phenotype characterizationin a large dystrophinopathic cohort with extended follow-up. J Neurol 2011;258:1610–23.

[15] Xie S, Lan Z, Qu N, et al. Detection of truncated dystrophin lacking the C-terminaldomain in a Chinese pedigree by next-generation sequencing. Gene 2012;499:139–42.

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