challenges and complexities in the interpretation of ... · challenges and complexities in the...

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

Thank you

Email contact:

steven.hardy@nuth.nhs.uk