Diagnostic mtDNA analysis Diagnostic mtDNA analysis in body fluids other than in body fluids other than

bloodblood

Sarah BallSarah Ball11, George Gray, George Gray11, Ros Quinlivan, Ros Quinlivan33, , Paul BrownPaul Brown44, Chris Hendriksz, Chris Hendriksz22

Birmingham Children’s Hospital, Birmingham Children’s Hospital, Departments of Clinical ChemistryDepartments of Clinical Chemistry11, Clinical , Clinical

IMDIMD22 and Neurology and Neurology33 and West Midlands and West Midlands Regional Molecular Genetics Service, Regional Molecular Genetics Service,

Birmingham Women's’ HospitalBirmingham Women's’ Hospital44

mtDNA disordersmtDNA disorders

Unusual genetic features:Unusual genetic features: Maternal inheritanceMaternal inheritance HeteroplasmyHeteroplasmy Variable levels of mutation load in Variable levels of mutation load in

different tissuesdifferent tissues

Samples for DNA testingSamples for DNA testing

Mutation level in DNA extracted from Mutation level in DNA extracted from blood may be below the level of blood may be below the level of detectabilitydetectability

If strong suspicion of mtDNA disease If strong suspicion of mtDNA disease testing lab will request DNA from a testing lab will request DNA from a biopsy of the affected tissue (e.g. liver, biopsy of the affected tissue (e.g. liver, kidney, muscle)kidney, muscle)

Tissue biopsy is expensive and invasiveTissue biopsy is expensive and invasive Difficulty reaching diagnosis when Difficulty reaching diagnosis when

appropriate samples are not availableappropriate samples are not available

Non-invasive samplesNon-invasive samples

West Midlands Inherited Metabolic West Midlands Inherited Metabolic Diseases Laboratory investigating use of Diseases Laboratory investigating use of non-invasive samples for testing for non-invasive samples for testing for mtDNA disordersmtDNA disorders

Urinary epithelium cells from 30ml early Urinary epithelium cells from 30ml early morning urine samplemorning urine sample

Buccal mucosal cells from saliva or buccal Buccal mucosal cells from saliva or buccal swabswab

DNA extracted from DNA extracted from urineurine

MW

mar

ker

Urin

e D

NA

1U

rine

DN

A 2

Urin

e D

NA

3

20kb +

Amplification of DNA Amplification of DNA extracted from urine: extracted from urine: m.3243A>G (MELAS) m.3243A>G (MELAS)

ampliconamplicon

MW

mar

ker

Urin

e 1

Urin

e 2

Urin

e 3

Urin

e 4

QC

sam

ple

No

DN

A c

ontr

ol

300bp PCR product

Amplification of DNA Amplification of DNA extracted from buccal extracted from buccal

swab: m.3243A>G (MELAS) swab: m.3243A>G (MELAS) ampliconamplicon

MW

mar

ker

Buc

cal s

crap

e D

NA

1

Buc

cal s

crap

e D

NA

2

QC

sam

ple

No

DN

A c

ontr

ol300 bp PCR product

Amplification of DNA Amplification of DNA extracted from urine: extracted from urine: m.8344A>G (MERRF) m.8344A>G (MERRF)

ampliconamplicon

MW

mar

ker

Urin

e D

NA

1

Urin

e D

NA

2

Urin

e D

NA

3

Urin

e D

NA

4

QC

sam

ple

No

DN

A c

ontr

ol100bp PCR product

Clinical samples to testClinical samples to test

Ready to test MELAS or MERRF Ready to test MELAS or MERRF mutations in saliva and urine mutations in saliva and urine

Awaiting samplesAwaiting samples

Analysis of m.8344A>GAnalysis of m.8344A>G

Index case: MKIndex case: MK 8 year old boy with muscle weakness8 year old boy with muscle weakness Blood DNA m.8344A>G mutation at a Blood DNA m.8344A>G mutation at a

high level of heteroplasmyhigh level of heteroplasmy Result consistent with a diagnosis of Result consistent with a diagnosis of

myoclonic epilepsy with ragged red myoclonic epilepsy with ragged red fibres (MERRF)fibres (MERRF)

Further studies on Further studies on m.8344A>Gm.8344A>G

MK’s mother, TK, has myoclonic MK’s mother, TK, has myoclonic epilepsyepilepsy

TK is needle phobic and had not had TK is needle phobic and had not had genetic testinggenetic testing

Following appropriate genetic Following appropriate genetic counselling obtained urine and saliva counselling obtained urine and saliva samples from MK and TKsamples from MK and TK

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 Analysis of m.8344A>G in MK and TKMK and TK

ResultsResults

Able to diagnose m.8344A>G Able to diagnose m.8344A>G mutation in urine and saliva samplesmutation in urine and saliva samples

Confirmed the diagnosis in the index Confirmed the diagnosis in the index casecase

Made a new diagnosis in his motherMade a new diagnosis in his mother In the index case mutation shows a In the index case mutation shows a

level of heteroplasmy in saliva and level of heteroplasmy in saliva and urine equal to or greater than the urine equal to or greater than the level in bloodlevel in blood

Further case 1Further case 1

Index case: JTIndex case: JT 38 year old female with muscle 38 year old female with muscle

weaknessweakness Blood DNA showed m.3243A>G Blood DNA showed m.3243A>G

mutation at very low level of mutation at very low level of heteroplasmy (~10%)heteroplasmy (~10%)

Urine and saliva samples showed Urine and saliva samples showed higher levels of heteroplasmyhigher levels of heteroplasmy

Further case 2Further case 2

Index case: AVIndex case: AV 8 year old boy with Leigh Syndrome8 year old boy with Leigh Syndrome Blood DNA showed m.9176T>C Blood DNA showed m.9176T>C

mutation at high level of mutation at high level of heteroplasmyheteroplasmy

Urine sample showed mutation at a Urine sample showed mutation at a very high level approaching very high level approaching homoplasmyhomoplasmy

Further workFurther work Further studies on patients with known mutations Further studies on patients with known mutations

are required to formally validate this approachare required to formally validate this approach

Need to compare urine and saliva mutation Need to compare urine and saliva mutation results with tissue biopsy specimens from the results with tissue biopsy specimens from the same patients to properly evaluate the use of same patients to properly evaluate the use of these specimens for testing for mtDNA mutationsthese specimens for testing for mtDNA mutations

We believe this is a useful addition to the test We believe this is a useful addition to the test repertoire of the laboratory testing for mtDNA repertoire of the laboratory testing for mtDNA mutations which may avoid unnecessary tissue mutations which may avoid unnecessary tissue biopsies for some patientsbiopsies for some patients