The investigation of

mitochondrial DNA
maintenance abnormalities
in adult onset
mitochondrial disease

Zarfishan Shabbir
Mitochondrial
Research Group,
Institute of Ageing
and Health &
Institute of Genetic
Medicine,
Newcastle
University
Nuclear mitochondrial interactions and Nuclear
genes involved in mtDNA maintenance

Nuclear genes encodes

signalling, genes involvedasin mtDNA
Nuclear transcriptional
replication/maintaining
well as replication factors. dNTP pools.
OXPHOS
Genes
5 highlighted
complexes in yellow
are
hexagoninare
involved theinvolved
production in MDS
of whilst
genes boxed in red are involved in
ATP.
mtDNAconstitute
They deletion disorders.
80 protein
Genes which
subunits,13 aresubunits
protein highlightedare in
yellow asby
encoded well as red(rest
mtDNA are are
associated
with both. (Courtesy
transcribed by nuclear DNA)
of Prof. Rob Taylor)
 Non-Neurological Mitochondrial Neurological
Respiratory Disease Optic neuropathy,
weakness optic atrophy,
retinitis pigmentosa

Cardiomyopathy Strokes, ataxia,

encephalopathy

Liver
Deafness
failure

Pancytopenia
↓ ATP Neuropathy
synthesis

Diabetes
Mellitus Myopathy, PEO,
Thyroid muscle pain
disease
 Features of the cohort
 Identify the genetic basis of mitochondrial disease in 35 adult German
patients with secondary mtDNA changes in muscle biopsies
 Diagnosed with CPEO, based on clinical, histochemical and biochemical
grounds
 Multiple mtDNA deletions which leads to COX-deficient muscle fibres are
found in association with mutations in candidate genes such as POLG,
POLG2, PEO1, RRM2B, TK2 and ANT1
1 2 3
1 2 3 4
- 9.9 kb
- 16.6 kb
multiple
mtDNA
deletions

CPEO COX-ve fibres Southern long-range PCR

 Methods
 Intronic primers
 Polymerase Chain Reaction for amplification of DNA
 Gel electrophoresis is used for the visualization of DNA
fragments
 ABI3130xl genetic analyser is used for sequencing
 Mutation Surveyor 3.2.3
 Reference Sequences from GenBank
 Alamut™v1.5 (Biosoftware).
 SIFT and GVGD
 Results
Sequencing of
POLG, PEO1 and
RRM2B revealed
11 intronic
variants, 11
known
pathogenic
mutations and 3
novel exonic
variants.

p.A467T (3 patients inp.POLG) p.R627Q (2 patients

V1044A (1 patient inp.M919L(novel,
POLG) in POLG)
1 patient in POLG)
p.W748S (1 patient in POLG) p.Y955C (1 patient in POLG)
p.F961S (1 patient in POLG) p. P587L (1 patient in POLG)
p. T251I (1 patient in POLG)
 Results
Sequencing of
POLG, PEO1 and
RRM2B revealed
11 intronic
variants, 11
known
pathogenic
mutations and 3
novel exonic
variants.

p. R186G (1 patient
p. R345p in RRM2B)
(1 patient in PEO1) p.p.T218I
F331L(novel,
(novel,
1 patient
1 patient
inin
RRM2B)
PEO1)
p. M455V (1 patient in PEO1)
p. M114R (1 patient in PEO1)
 Results for familial samples in RRM2B
 Patient SW_03 had a novel heterozygous missense change (p.Thr218Ile) and known heterozygous
p.Arg186Gly mutation.
p.T218I found in Patient’s sister II
p.R186G found in Patient’s father and Sister I
 Discussion and Conclusion
 Results of this study corresponds with patients phenotypes and affirm that multiple
mitochondrial deletions in the nuclear genes of the patients lead to severe mitochondrial
disorders.
Patients in the cohort
present the symptoms of
CPEO, neuropathy and
ptosis.
POLG gene is the most
common gene for
mitochondrial mutations
p.A467T is commonly
found in Norwegian and
British population
p.W748S is commonly
found in Finnish
population
p.T251L is commonly
found in cis with p.P587L,
both are usually
heterozygous recessive
mutations but they are
found as homozygous.
 Limitations and further analysis
 ABI3130xI genetic analyser Looks for point mutations, small
deletions or insertions.
 MLPA (Multiple Ligation-dependent Probe Amplification) can
find most large deletions, and many duplications.
 Novel changes in POLG gene p.Met919Leu and RRM2B gene
p.THr218Ile are predicted to affect protein function.
 POLG2 and ANT1 sequencing was not completed hence it is
important to look for mutations in these genes for all the
undiagnosed patients.
 Determine the pattern of inheritance through familial sample
sequencing.
 Exome Sequencing for selective sequencing the coding
regions – in a cheaper way.
 Acknowledgment
I would like to thank Professor Rob Taylor for his guidance &
Dr. Marcus Deschauer, University of Halle, Germany for providing
us with patient samples.
I would also like to thank Charlotte Alston for her assistance and
support in the lab
Lastly, I am grateful to the mitochondrial diagnostic team for
their help.

References
www.uib.no/imagearchive/storthovedtekstbilde_coxsdh1
70605BJN.jpg
http://tools.niehs.nih.gov/polg/
http://www.ncbi.nlm.nih.gov/pubmed/17725985
http://genetics.bwh.harvard.edu/pph/