Becker muscular dystrophy patients

Neuromuscular Disorders 18 (2008) 667–670 www.elsevier.com/locate/nmd

MLPA analysis/complete sequencing of the DMD gene in a group of Bulgarian Duchenne/Becker muscular dystrophy patients Albena Todorova a,b,*, Tihomir Todorov a,b, Bilyana Georgieva a, Michaela Lukova b, Velina Guergueltcheva c, Ivo Kremensky d, Vanyo Mitev a a

Department of Chemistry and Biochemistry, Medical Faculty, Sofia Medical University, 2 ‘‘Zdrave” str., Sofia 1431, Bulgaria b Genetic Madico-Diagnostic Laboratory ‘‘Genica”, Sofia, Bulgaria c Clinic of Neurology, Alexandrovska Hospital, Sofia Medical University, Sofia, Bulgaria d National Genetic Laboratory, Hospital of Obstetrics and Gynecology, Sofia Medical University, Sofia, Bulgaria Received 25 February 2008; received in revised form 20 May 2008; accepted 23 June 2008

Abstract Duchenne/Becker muscular dystrophy (DMD/BMD), the most common X-linked muscular dystrophy is caused by mutations in the enormously large DMD gene, encoding the protein called dystrophin. This gene was screened in a group of 27 unrelated Bulgarian DMD/BMD patients by MLPA analysis/complete sequencing. We managed to clarify the disease-causing mutation in 96.3% of the analyzed families. The MLPA analysis revealed 17 deletions (including a deletion of the very last exon 79), 6 duplications and 1 point mutation. Two additional point mutations (one of them novel) were detected after complete sequencing of the DMD gene. Altogether, 25 carriers and 11 noncarriers were detected in our families. The MLPA test proved to be a powerful tool in detecting deletions/duplications and in some cases point mutations/polymorphisms along the DMD gene. Using this approach in combination with a direct gene sequencing a number of Bulgarian DMD/BMD patients are genetically clarified and prepared for gene therapy in future. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Duchenne/Becker muscular dystrophy; DMD gene; Dystrophin; MLPA; Deletions; Duplications; Point mutations

1. Introduction Duchenne/Becker muscular dystrophy (DMD/BMD; OMIM#310200; 300376), the most common X-linked muscular dystrophy is caused by mutations in the enormously large DMD gene, encoding the protein called dystrophin. About 60% of the cases are due to deletions of one or more exons, in 20% duplications of one or more exons are detected, while point mutations of different type and location are responsible for the remaining 10% of the cases [1–3]. The detection of the duplicated regions along the DMD gene was technically complicated until now. The *

Corresponding author. Department of Chemistry and Biochemistry, Medical Faculty, Sofia Medical University, 2 ‘‘Zdrave” str., Sofia 1431, Bulgaria. Tel.: +359 2 953 07 15; fax: +359 2 954 17 15. E-mail address: [email protected] (A. Todorova). 0960-8966/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.06.369

same was true for carrier status determination, where mainly indirect analyses by highly informative polymorphic markers, were routinely used and some attempts for semi-quantitative direct tests were offered [4,5]. Recently, the designed for research use MLPA (multiplex ligationdependent probe amplification) test [6] was successfully applied also for analysis of the DMD gene, which solved a lot of complications in routine diagnostic approaches offered to DMD/BMD families. Here, we present our results and experience in applying the combined MLPA test/direct sequencing of the DMD gene for diagnostic purposes in Bulgaria. 2. Materials and methods The SALSA MLPA P034/P035 kit [6] was applied exactly as recommended by the manufacturers in a group

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Table 1 Mutations detected in the group of Bulgarian DMD/BMD patients, some clinical data and clarified carrier status in affected families Diagnosis

1. DMD

Mutation**/reading frame

CK

Clinical symptoms

Family history

First walk (months)

Age at first symptoms (years)

Special clinical findings

Family members tested Patients

Carriers

Noncarriers

ex48ex50dup ->c.6913?_7309+?dup out-of-frame ex02ex33dup -> c.32?_4674+?dup out-of-frame

4500

14

3

Lordosis

Yes

1

6

1

1521

29

1

No

1

1

1

3. DMD

ex08ex11dup ->c.650?_1331+?dup out-of-frame

9000

15

5

No

1

4. IMD de novo 5. BMD

ex49ex50del ->c.7099?_7309+?del out-of-frame ex13ex40dup ->c.1483?_5739+?dup in-frame ex42ex45del ->c.5923?_6614+?del out-of-frame ex44ex44del ->c.6291?_6438+?del out-of-frame ex58ex58del ->c.8548?_8668+?del out-of-frame

9212

13

9

No

1

1107

12

12

At 10 months cannot sit, twist, keep the head upright Walking on toes, cannot climb stairs alone Scapula alata, lumbal lordosis Profound lordosis

Yes

1

High

14

2

nd

No

1

12460

14

2

Muscle hypotony

No

1

14548

14

5

No

1

ex55ex55del ->c.8028?_8217+?del out-of-frame ex79ex79del->c.11047?_11064+?del does not encode a functional protein? ex53ex54del ->c.7661?_8027+?del out-of-frame ex02ex10dup ->c.32?_1149+?dup out-of-frame ex46ex52del ->c.6615?_7660+?del out-of-frame ex45ex47del ->c.6439?_6912+?del in-frame Exon 23 c.2991C>G, p.Tyr997X ex46ex50del ->c.6615?_7309+?del out-of-frame ex44ex44del ->c.6291?_6438+?del out-of-frame Exon 59 c.8776C>T, p.Gln2926X* ex45ex54del ->c.6439?_8027+?del out-of-frame ex45ex48del ->c.6439?_7098+?del in-frame ex45ex52del ->c.6439?_7660+?del out-of-frame Exon 7 c.583C>T, p.Arg195X Ex08ex11del->c.650?_1331+?del out-of-frame

5979

13

3

Scapula alata, lumbal lordosis, contractures of Achilles tendons nd

No

1

14390

13

6

Muscle hypotony, profound lordosis

No

1

High

14

3

nd

Yes

1

2

409

15

3

Wheelchair bound

Yes

1

1

Yes

–

1

2. DMD

6. DMD 7. DMD 8. DMD

9. DMD 10. DMD

11. DMD 12. DMD 13. DMD 14. BMD 15. DMD 16. DMD de novo 17. DMD 18. DMD 19. DMD de novo 20. BMD 21. DMD 22. DMD 23. DMD

24. DMD 25. DMD 26. DMD

ex48ex50del ->c.6913?_7309+?del out-of-frame ex51ex51dup ->c.7310?_7542+?dup out-of-frame ex45ex50del ->c.6439?_7309+?del out-of-frame

No index patient

1 1

12000

12

11

Elbow contractures

No

1

1

17100

13

3

Died at age 17

No

1

3

3982

14

2

nd

No

1

2280

14

3

No

1

1

9000

15

2

Yes

1

1

15395

16

2

Profound lumbal lordosis, scapula alata Hyporeflexia in lower limbs Lordosis

No

1

800

12

13

Muscle hypotony

No

1

420

14

3

Wheelchair bound

Yes

1

12000

16

2

No

1

1

18352

12

4

Hyporeflexia in Achilles and knees Lumbal lordosis, scapula alata, Achilles contractures

No

1

2

Yes

–

1

Yes

1

1

No

–

1

No index patient 4500

16

No index patient

3

Wheelchair bound

1

1

2 1

1 1

1

1

**The mutation nomenclature is according to www.dmd.nl; IMD, intermediate muscular dystrophy; * novel mutation; nd, no data; –, no index patient.

A. Todorova et al. / Neuromuscular Disorders 18 (2008) 667–670

of 27 unrelated Bulgarian DMD/BMD families with unclear molecular defect. Patients were clinically diagnosed as DMD/BMD only on the base of clinical symptoms, family history, EMG data and creatine kinase levels. Three families, in which the patient was not available for the study, but sisters requested to know their carrier status, were also included in the analysis. The patients were tested by MLPA in separate groups by gender, including three normal controls in each sample. The electrophoretic separation was performed on ABI 310 genetic analyzer (Applied Biosystems). The obtained data was analyzed by Coffalyser [6] and calculations in Excel (data not shown) and interpreted afterwards. The entire coding sequence or defined exons of the DMD gene, including intron/exon borders were directly sequenced in some cases. 3. Results The presented group consists of 19 DMD, 3 BMD and 1 IMD (intermediate muscular dystrophy) affected boys. The first symptoms in all of them have been very similar: walking on toes, difficulties in climbing stairs, running and uprising. Some special clinical findings are presented in Table 1. The CK (creatine kinase) levels were highly elevated in all the cases, except the handicapped ones. The main group of patients started walking between 12 and 16 months, with one exception #2 – at 2 years and 5 months. The clinical manifestation of the disease in this boy was drastically severe (Table 1).

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All available index patients (23), but one (#14 in Table 1) was pre-screened for deletions in the DMD gene by standard multiplex set of primers. Patient #14 has been clinically diagnosed as Emery-Dreifuss muscular dystrophy (OMIM#181350; OMIM#310300), severe contractures were presented in this case from very early disease stage. He was included in the study by chance, to look for a precise diagnosis, as Emery-Dreifuss muscular dystrophy (X-linked and autosomal-dominant) has not been genetically confirmed. In patient #4 the deletion of exons 49–50 was detected by multiplex PCR, but the family requested confirmation with alternative method. In 6 cases (#6, #16, #19, #20, #21 and #23) the deletions were detected by multiplex PCR, but deletion borders were not precisely determined, or carrier status determination in these families required MLPA. In two cases (#7 and #17), the deletion of exon 44 failed to be detected by multiplex PCR. The obtained fragments for exon 44 in the standard multiplex set in both cases were very weak, most probably due to unspecific amplification. These cases were considered as no deletions. The remaining 14 index patients were classified as unknown mutations after multiplex PCR. The MLPA analysis revealed 17 deletions (3 de novo) and 6 duplications in our sample group (Table 1 and Fig. 1). DMD gene mutations were detected in 96.3% (26/27) of the tested families. Considering the localization and size of the deleted fragments, detected in the present sample, it is interesting to mention the deletion of the very last exon of the DMD gene

Fig. 1. Capillary electrophoresis pattern in a patient with duplication of exons 48–50 in comparison to a control sample. Each patient sample was analyzed in two subsequent electrophoretic runs.

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(exon 79), detected in a DMD boy. It is not clear where the deletion ends, but the clinical picture in our case is compatible with DMD phenotype (early onset and fast progression). A similar mutation has been described earlier in a BMD patient as a part of a contiguous gene deletion syndrome [7]. Regarding the size of the duplicated fragments presented here, it is interesting to point that two duplications of exons 2–33 and 13–40 cover more than 1/3 of the gene sequence, resulting in DMD and BMD phenotype, respectively. Most probably, the duplications situated close to the N-terminus of the dystrophin are associated with severe phenotype, while the ones shifted along the rod-domain provoke weaker BMD phenotype. All deletions, detected by MLPA were confirmed by single exon PCR amplifications. Both exon 44 deleted cases (#7 and #17) were confirmed by newly designed primers situated close to the exon/intron borders and not by the utilized ones in the multiplex set, situated deeply in the intronic sequence. In one patient, the exon 23 was missing on the MLPA electropherogram, but the single amplification of exon 23 showed its presence in the patient. Subsequently, point mutation c.2991C?G, p.Tyr997X was detected after sequencing of the exon. Three patents tested negative by MLPA were sequenced for the entire coding sequence of the DMD gene. Two point mutations were detected after sequencing: c.8776C?T, p.Gln2926X (novel mutation) and c.583C?T, p.Arg195X. Only one patient after complete analysis of the DMD gene was proved not to have disease-causing changes in this gene and the clinical diagnosis of BMD need to be revised. Immunohistochemical analysis on muscular biopsy of this patient pointed as a second most probable diagnosis calpainopathy (OMIM#253600), as calpain 3 had not been detected in the sample. In fact, the less severe clinical symptoms in this case, as well as creatine kinase levels, are much more compatible with calpainopathy. The MLPA analysis was also applied in three families (#13, #24 and #26) where no index patient was available. We identified three deletions in these families and two sisters of affected boys were genetically proved to be noncarriers. Altogether, we detected 25 carriers and 11 noncarriers in our families (Table 1). 4. Discussion Our study proved that the combination of MLPA test with direct sequencing of the entire DMD gene could be a powerful diagnostic tool in DMD/BMD families. Using this diagnostic approach we managed to clarify the disease-causing mutation in 96.3% of the analyzed families and to estimate precisely the deletion/duplication borders in all the cases. The classical multiplex PCR of 18 exons in the deletion hot-spot region of the DMD gene [8,9] could partially

detect 10 deletions (59% of deletions) in the present sample, only when the index patient is available for the study. This means 37% of the presented families to be genetically assessed. On the other hand, the presented here MLPA analysis was successful in families with no index patient. Moreover, the MLPA data is easily interpreted for direct carrier status determination. The detected deletions in our group account for 65% of the mutations, the duplications – 23% and the point mutations – 12%. Although, the MLPA tests were designed for research use [6], our experience reveals that the DMD/BMD kit could be successfully used for diagnostic purposes. It proved to be a powerful tool in detecting deletions/duplications along the DMD gene. In some cases point mutations/ polymorphisms could also be detected, if they affect the hybridization/ligation site resulting in fragment loss. This requires each single exon failure to be proved by single exon amplification. In case the fragment appears, it has to be sequenced to find out the change along the sequence, which provoked the fragment loss in MLPA. Using the MLPA approach in combination with a direct gene sequencing a number of Bulgarian DMD/BMD patients are genetically clarified and prepared for gene therapy in future. Acknowledgments The fellowship of the Alexander von Humboldt Foundation to Dr. Albena Todorova is gratefully acknowledged. The study was supported by the Grant No. 17/ 2007 and the Grant No. 24/2008 Sofia Medical University. References [1] DMD Genetic Therapy Group. Leiden muscular dystrophy pages 1997. Available from: http://www.dmd.nl. [2] 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(2):135–44. [3] White SJ, Aartsma-Rus A, Flanigan KM, Weiss RB, Kneppers AL, Lalic T, et al. Duplications in the DMD gene. Hum Mutat 2006;27(9):938–45. [4] Bronzova J, Todorova A, Kalaydjieva L. Detection of carriers of deletions in the dystrophin gene in Bulgarian DMD–BMD families. Hum Genet 1994;93:170–4. [5] Miorin M, Todorova A, Vitiello L, Rosa M, Mostacciuolo ML, Danieli GA. Detection of heterozygotes for intragenic deletions in families with recurrence of Duchenne or Becker muscular dystrophy. Basic Appl Myol 1997;7(3):265–9. [6] MRC Holland, Amsterdam. Available from: http://www.mlpa.com. [7] Greener MJ, Sewry CA, Muntoni F, Roberts RG. The 30 -untranslated region of the dystrophin gene – conservation and consequences of loss. Eur J Hum Genet 2002;10(7):413–20. [8] Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 1988;16:11141–56. [9] Beggs AH, Koenig M, Boyce FM, Kunkel LM. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 1990;86:45–8.