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Challenges and complexities in the interpretation of mitochondrial DNA
(mtDNA) variants
Steven Hardy
NHS Highly Specialised Service for Rare Mitochondrial Disorders, Newcastle upon Tyne
“Mitochondrial disease” a collective term for many different clinical disorders united by a failure
of mitochondrial function and energy production
“…..Any organ, at any age…….”
Nuclear DNA: 3,000,000,000 bp
(>20,000 genes; ~1300+ proteins)
mtDNA: 16,569 bp
(37 genes; 13 proteins)
Lightowlers et al. 2015. Science, 349:1494-1498.
Nuclear DNA: 3,000,000,000 bp
(>20,000 genes; ~1300+ proteins)
mtDNA: 16,569 bp
(37 genes; 13 proteins)
Lightowlers et al. 2015. Science, 349:1494-1498.
Maternal inheritance
Heteroplasmy
Threshold effect
Mitotic segregation
mtDNA – genetic rules
>1500 mtDNA genomes sequence in Newcastle
Highly polymorphic:
>3,300 benign polymorphisms and >275 pathogenic variants listed on
MITOMAP database
5 - >50 variants per individual
20% individuals harbour a variant of uncertain clinical
significance
Whole mitochondrial genome sequencing
200x minimum coverage
“Interpretation of mitochondrial variants other than well established pathogenic
variants is complex and remains challenging… giving the difficulty in assessing
mitochondrial variants, a separate evidence checklist has not been included”
“… because many mitochondrial variants are misssense variants, evidence
criteria for truncating variants likely will not be helpful”
“… functional studies are not readily available”
“ Frequency data and published studies may often be the
only assessable criteria on the checklist”
mtDNA variant classification: canonical criteria
Consensus species panel established
for mt-tRNA variants (Yarham et al.,
2011); not yet for protein-encoding
variants
Database interrogation
(https://www.mitomap.org//MITOMAP)
Familial segregation studies
(matrilineal)
Tissue segregation studies (higher in
post-mitotic tissue e.g. SKM; lower in
mitotic tissue e.g. blood)
Single muscle fibre segregation studies
Quantitative assay e.g. NGS or
pyrosequencing
McFarland et al. 2004. Trends Genet., 20(12), 591-596
Our pipeline – case studyPatient presented with myopathic phenotype
0
20
40
60
80
100
120
Complex I ComplexII
ComplexIII
ComplexIV
Control
Patient** *
A B
C
% e
nzym
e a
ctivity
Lines of evidence
Patient
2. Predicted effects on mt-tRNAPro structure and conservation analysis
1. Database interrogation
DHU Loop
TΨC Loop
Aminoacyl acceptor
stem
Anticodon Loop
m.15998A>T
Lines of evidence3. Segregation patterns
Tissue Patient Mum
SKM 85% Not tested
Blood n.d. n.d.
Buccal n.d. n.d.
Urine n.d. n.d.
4. Single muscle fibre segregation studies
Heteroplasmy levels (%)
COX-deficient fibres COX-positive Fibres
92.47 ± 0.62 (n=15) 38.33 ± 6.09 (n=12)
p<0.0001
Hardy et al. 2016. Neurol Genet, 2(4), e82
Homoplasmic mtDNA variants
Cardiac
SKM
McFarland et al. 2002. Nat genet, 30, 145-146
mt-tRNAVal
m.1624C>T
tRNA Variant Clinical phenotype Decreased steady-state tRNA
levels in affected tissue
Val m.1624C>T Fatal infantile lactic
acidosis, Leigh
Syndrome
Cys m.5618A>G Epilepsy, dystonia
Ile m.4300A>G Isolated hypertrophic
cardiomyopathy
Glu m.14709T>C Myopathy with diabetes
mt-tRNACys
mt-tRNALeu(UUR)C Pat Pat C
Pathogenic homoplasmic mtDNA variants
Discordance with functional studies
MTATP6 m.8839G>C p.(Ala105Pro) detected
Patient presented with NARP phenotype (strong prior likelihood of MTATP6/8 pathogenic
variant)Blood n.d.
Blood n.d
Urine n.d.
Blood n.d
Urine n.d.
SKM 58%
Urine 76%
Blood 26%
BUT
no evidence of complex V deficiency
Summary• mtDNA variants of uncertain clinical significance continue to be detected –
likely to increase with inclusion of mitochondrial genes on GeL panels
• Variant interpretation complicated by unique genetics and extensive
clinical/genetic heterogeneity
• Canonical criteria for classification is helpful but by no means fully
prescriptive
• Multidisciplinary approach is key – need to link all findings together
• Robust classification is crucial to inform reproductive options
