Diagnostic mtDNA analysis in body fluids other than blood.pptVIP

Diagnostic mtDNA analysis in body fluids other than blood Sarah Ball1, George Gray1, Ros Quinlivan3, Paul Brown4, Chris Hendriksz2 Birmingham Children’s Hospital, Departments of Clinical Chemistry1, Clinical IMD2 and Neurology3 and West Midlands Regional Molecular Genetics Service, Birmingham Womens’ Hospital4 mtDNA disorders Unusual genetic features: Maternal inheritance Heteroplasmy Variable levels of mutation load in different tissues Samples for DNA testing Mutation level in DNA extracted from blood may be below the level of detectability If strong suspicion of mtDNA disease testing lab will request DNA from a biopsy of the affected tissue (e.g. liver, kidney, muscle) Tissue biopsy is expensive and invasive Difficulty reaching diagnosis when appropriate samples are not available Non-invasive samples West Midlands Inherited Metabolic Diseases Laboratory investigating use of non-invasive samples for testing for mtDNA disorders Urinary epithelium cells from 30ml early morning urine sample Buccal mucosal cells from saliva or buccal swab DNA extracted from urine Amplification of DNA extracted from urine: m.3243AG (MELAS) amplicon Amplification of DNA extracted from buccal swab: m.3243AG (MELAS) amplicon Amplification of DNA extracted from urine: m.8344AG (MERRF) amplicon Clinical samples to test Ready to test MELAS or MERRF mutations in saliva and urine Awaiting samples Analysis of m.8344AG Index case: MK 8 year old boy with muscle weakness Blood DNA m.8344AG mutation at a high level of heteroplasmy Result consistent with a diagnosis of myoclonic epilepsy with ragged red fibres (MERRF) Further studies on m.8344AG MK’s mother, TK, has myoclonic epilepsy TK is needle phobic and had not had genetic testing Following appropriate genetic counselling obtained urine and saliva samples from MK and TK Analysis of m.8344AG in MK and TK Results Able to diagnose m.8344AG mutation in urine and saliva samples Confirmed the diagnosis in the index case Made a new diagnosi