1. Diagnostic mtDNA analysis in body fluids other than blood Sarah Ball 1, George Gray 1, Ros Quinlivan 3, Paul Brown 4, Chris Hendriksz 2 Birmingham Children’s Hospital, Departments of Clinical Chemistry 1, Clinical IMD 2 and Neurology 3 and West Midlands Regional Molecular Genetics Service, Birmingham Women's’ Hospital 4

2. mtDNA disorders Unusual genetic features: Maternal inheritance Maternal inheritance Heteroplasmy Heteroplasmy Variable levels of mutation load in different tissues Variable levels of mutation load in different tissues

3. Samples for DNA testing Mutation level in DNA extracted from blood may be below the level of detectability 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) 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 Tissue biopsy is expensive and invasive Difficulty reaching diagnosis when appropriate samples are not available Difficulty reaching diagnosis when appropriate samples are not available

4. Non-invasive samples West Midlands Inherited Metabolic Diseases Laboratory investigating use of non-invasive samples for testing for mtDNA disorders 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 Urinary epithelium cells from 30ml early morning urine sample Buccal mucosal cells from saliva or buccal swab Buccal mucosal cells from saliva or buccal swab

5. DNA extracted from urine MW marker Urine DNA 1 Urine DNA 2 Urine DNA 3 20kb +

6. Amplification of DNA extracted from urine: m.3243A>G (MELAS) amplicon MW marker Urine 1 Urine 2 Urine 3 Urine 4 QC sample No DNA control 300bp PCR product

7. Amplification of DNA extracted from buccal swab: m.3243A>G (MELAS) amplicon MW marker Buccal scrape DNA 1 Buccal scrape DNA2 QC sample No DNA control 300 bp PCR product

8. Amplification of DNA extracted from urine: m.8344A>G (MERRF) amplicon MW marker Urine DNA 1 Urine DNA 2 Urine DNA 3 Urine DNA 4 QC sample No DNA control 100bp PCR product

9. Clinical samples to test Ready to test MELAS or MERRF mutations in saliva and urine Ready to test MELAS or MERRF mutations in saliva and urine Awaiting samples Awaiting samples

10. Analysis of m.8344A>G Index case: MK Index case: MK 8 year old boy with muscle weakness 8 year old boy with muscle weakness Blood DNA m.8344A>G mutation at a high level of heteroplasmy Blood DNA m.8344A>G mutation at a high level of heteroplasmy Result consistent with a diagnosis of myoclonic epilepsy with ragged red fibres (MERRF) Result consistent with a diagnosis of myoclonic epilepsy with ragged red fibres (MERRF)

11. Further studies on m.8344A>G MK’s mother, TK, has myoclonic epilepsy MK’s mother, TK, has myoclonic epilepsy TK is needle phobic and had not had genetic testing TK is needle phobic and had not had genetic testing Following appropriate genetic counselling obtained urine and saliva samples from MK and TK Following appropriate genetic counselling obtained urine and saliva samples from MK and TK

12. 100bp 1 2 3 4 5 6 7 8 1 MK venous blood 2 MK urine 3 MK saliva 4 TK urine 5 TK saliva 6 mutation absent QC sample 7 mutation present QC sample 8 no template control 100bp 73bp Analysis of m.8344A>G in MK and TK

13. Results Able to diagnose m.8344A>G mutation in urine and saliva samples Able to diagnose m.8344A>G mutation in urine and saliva samples Confirmed the diagnosis in the index case Confirmed the diagnosis in the index case Made a new diagnosis in his mother Made a new diagnosis in his mother In the index case mutation shows a level of heteroplasmy in saliva and urine equal to or greater than the level in blood In the index case mutation shows a level of heteroplasmy in saliva and urine equal to or greater than the level in blood

14. Further case 1 Index case: JT Index case: JT 38 year old female with muscle weakness 38 year old female with muscle weakness Blood DNA showed m.3243A>G mutation at very low level of heteroplasmy (~10%) Blood DNA showed m.3243A>G mutation at very low level of heteroplasmy (~10%) Urine and saliva samples showed higher levels of heteroplasmy Urine and saliva samples showed higher levels of heteroplasmy

15. Further case 2 Index case: AV Index case: AV 8 year old boy with Leigh Syndrome 8 year old boy with Leigh Syndrome Blood DNA showed m.9176T>C mutation at high level of heteroplasmy Blood DNA showed m.9176T>C mutation at high level of heteroplasmy Urine sample showed mutation at a very high level approaching homoplasmy Urine sample showed mutation at a very high level approaching homoplasmy

16. Further work Further studies on patients with known mutations are required to formally validate this approach Further studies on patients with known mutations are required to formally validate this approach Need to compare urine and saliva mutation results with tissue biopsy specimens from the same patients to properly evaluate the use of these specimens for testing for mtDNA mutations Need to compare urine and saliva mutation results with tissue biopsy specimens from the same patients to properly evaluate the use of these specimens for testing for mtDNA mutations We believe this is a useful addition to the test repertoire of the laboratory testing for mtDNA mutations which may avoid unnecessary tissue biopsies for some patients We believe this is a useful addition to the test repertoire of the laboratory testing for mtDNA mutations which may avoid unnecessary tissue biopsies for some patients