Duchenne Muscular Dystrophy: The Diagnostic Process

1. Duchenne Muscular Dystrophy:The Diagnostic Process

2. Care at Diagnosis • Aim: provide accurate diagnosis as quickly as possible when DMD is suspected • Allow the doctor and family to plan for care • Inform family about course of disease and treatment options • Provide family with appropriate care, including genetic counselling, ongoing support and education

3. Care at Diagnosis 2 • Ideally performed by specialist neuromuscular doctor • Able to provide clinical assessment, and initiate/interpret further investigations • Post-diagnosis family follow up and support often supplemented by genetic counsellors • Families should be offered genetic counselling • Contact with support/advocacy group can be helpful

4. When to suspect DMD Without family history of DMD, suspicions raised by: • Problems with muscle function • High levels of CreatineKinase (CK) • High levels of transanimases AST/ALT

5. Problems with muscle function • Often noticed by family • Symptoms include: • Late walking • Trouble running, jumping or climbing stairs • Falls easily • Enlarged calf muscles (hypertrophy) • May have a tendency to walk on their toes • May have a speech delay • Gower’s manoeuvre

6. Calf hypertrophy • [Image]

7. Toe-walking • [Image and/or video]

8. Gower’s Manouvre • [Video]

9. High levels of CK in blood test • Levels typically > 10-100+ times normal range • Should prompt an urgent referral to a neuromuscular specialist • Not alone sufficient to confirm DMD • Also seen in other muscle conditions, e.g. various forms of limb-girdle muscular dystrophy (LGMD) • “High CK, don’t delay”

10. High levels of AST/ALT • High levels of liver enzymes in blood tests • Aspartateaminotransferase (AST) • Alanine aminotransferase(ALT) • May be detected on routine blood screening • Often associated with liver disease, but can be caused by muscular dystrophies • Should raise suspicion of high CK and thus muscular dystrophy • Liver biopsy is not recommended until CK checked unless overt liver disease is present

11. Confirming the Diagnosis • DMD caused by dystrophin mutation at Xp21 • Diagnosis should be confirmed by genetic testing • Other tests sometimes performed as well, most notably muscle biopsies

12. Genetic Testing • Genetic testing always necessary even if DMD first suggested by muscle biopsy • Gives specific, detailed information on mutation • Helps determine mutation-specific trial eligibility • Assists family with decisions on carrier status, pre-natal diagnosis and further pregnancies • Full characterisation (deletion endpoints, exact position of point-mutations) is also required • Allows correlation the predicted effect of the mutation on the reading frame of the gene • Major determinant of phenotypic variability

13. Types of Genetic Test • Multiplex PCR • Widely available, least expensive • Only detects deletion, doesn’t cover whole gene • Multiplex ligation-dependent probe amplification (MLPA) • Now in common use: covers whole gene, detects deletions and duplications and allows carrier testing • Others: • Amplifiable probe hybridisation • Single-condition amplification/internal primer • Detects deletions and provides sequence data

14. Importance of Carrier Testing • If mother is a carrier, other children could be at risk of developing DMD • Additional possibility of germlinemosaicism • Carrier status important for female family on mother’s side, who should also be offered testing • Small risk to carriers of developing weak heart or leg weakness in later life. Knowledge of carrier status allows appropriate monitoring.

15. Muscle Biopsy Analysis • Analyses amount of dystrophin in muscle cells • If diagnosis already confirmed by genetic testing, may be unnecessary • If diagnosis is made by biopsy, genetic testing still essential to determine specific mutation • Types of biopsy • Open muscle biopsy – if DMD is not the only possible diagnosis • Needle biopsy – appropriate if testing is only for DMD, or clinician skilled in taking multiple cores of tissue from paediatric patients • Conchotome technique preferred where available • Two tests necessary: immunocytochemistry and immunoblotting for dystrophin. Should be performed by an experienced neuromuscular pathologist

16. Immunocytochemistry • Staining performed on muscle biopsy sections • More likely to be available in some areas than immunoblotting for dystrophin • [Images showing normal/DMD cases from muscle cells]

17. Immunoblotting • Blotting of homogenised tissue sample • Less likely to be available than immunocytochemistry in some areas • [Images showing normal/DMD cases from blotting]

18. Other Tests • Previously, other tests were used in diagnosis • Electromyography (EMG) • Nerve conduction • These are not appropriate nor necessary for the evaluation of DMD • Electron microscopy is not required to confirm DMD

19. References & Resources • The Diagnosis and Management of Duchenne Muscular Dystrophy, Bushby K et al, Lancet Neurology 2010 9 (1) 77-93 & Lancet Neurology 2010 9 (2) 177-189 • Particularly references, p186-188 • Best Practice Guidelines on molecular diagnostics in Duchenne/Becker muscular dystrophies, Abbs S et al, Neuromuscular Disorders 20 (2010) 422-427 • The Diagnosis and Management of Duchenne Muscular Dystrophy: A Guide for Families • TREAT-NMD website: www.treat-nmd.eu • CARE-NMD website: www.care-nmd.eu