Duchenne Muscular Dystrophy: The Diagnostic Process

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

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

When to suspect DMD Without family history of DMD, suspicions raised by: 1.Problems with muscle function 2.High levels of Creatine Kinase (CK) 3.High levels of transanimases AST/ALT

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

Calf hypertrophy [Image]

Toe-walking [Image and/or video]

Gower’s Manouvre [Video]

High levels of CK in blood test Levels typically > 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”

High levels of AST/ALT High levels of liver enzymes in blood tests – Aspartate aminotransferase (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

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

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

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

Importance of Carrier Testing If mother is a carrier, other children could be at risk of developing DMD Additional possibility of germline mosaicism 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.

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

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]

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

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

References & Resources The Diagnosis and Management of Duchenne Muscular Dystrophy, Bushby K et al, Lancet Neurology (1) & Lancet Neurology (2) – Particularly references, p Best Practice Guidelines on molecular diagnostics in Duchenne/Becker muscular dystrophies, Abbs S et al, Neuromuscular Disorders 20 (2010) The Diagnosis and Management of Duchenne Muscular Dystrophy: A Guide for Families TREAT-NMD website: CARE-NMD website: