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 Women's’ 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
20kb +
No DNA control
QC sample
Urine 4
Urine 3
Urine 2
Urine 1
MW marker
Amplification of DNA extracted
from urine: m.3243A>G
(MELAS) amplicon
300bp PCR product
No DNA control
QC sample
Buccal scrape DNA2
Buccal scrape DNA 1
MW marker
Amplification of DNA extracted
from buccal swab: m.3243A>G
(MELAS) amplicon
300 bp PCR product
No DNA control
QC sample
Urine DNA 4
Urine DNA 3
Urine DNA 2
Urine DNA 1
MW marker
Amplification of DNA extracted
from urine: m.8344A>G
(MERRF) amplicon
100bp PCR
product
Clinical samples to test


Ready to test MELAS or MERRF mutations
in saliva and urine
Awaiting samples
Analysis of m.8344A>G




Index case: MK
8 year old boy with muscle weakness
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)
Further studies on m.8344A>G



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.8344A>G in MK
and TK
100bp
1
2
3
4
5
6
7
8
100bp
73bp
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
Results




Able to diagnose m.8344A>G mutation in
urine and saliva samples
Confirmed the diagnosis in the index case
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
Further case 1




Index case: JT
38 year old female with muscle weakness
Blood DNA showed m.3243A>G mutation
at very low level of heteroplasmy (~10%)
Urine and saliva samples showed higher
levels of heteroplasmy
Further case 2




Index case: AV
8 year old boy with Leigh Syndrome
Blood DNA showed m.9176T>C mutation
at high level of heteroplasmy
Urine sample showed mutation at a very
high level approaching homoplasmy
Further work



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
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