Challenges and complexities in the interpretation of mitochondrial DNA (mtdna) variants

Transcription

1 Challenges and complexities in the interpretation of mitochondrial DNA (mtdna) variants Steven Hardy NHS Highly Specialised Service for Rare Mitochondrial Disorders, Newcastle upon Tyne

2 Mitochondrial disease a collective term for many different clinical disorders united by a failure of mitochondrial function and energy production

3 ..Any organ, at any age.

4 mtdna: 16,569 bp (37 genes; 13 proteins) Nuclear DNA: 3,000,000,000 bp (>20,000 genes; ~1300+ proteins) Lightowlers et al Science, 349:

5 mtdna: 16,569 bp (37 genes; 13 proteins) Nuclear DNA: 3,000,000,000 bp (>20,000 genes; ~1300+ proteins) Lightowlers et al Science, 349:

6 mtdna genetic rules Maternal inheritance Heteroplasmy Threshold effect Mitotic segregation

7 Whole mitochondrial genome sequencing 200x minimum coverage >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

8 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

9 mtdna variant classification: canonical criteria Consensus species panel established for mt-trna variants (Yarham et al., 2011); not yet for protein-encoding variants Quantitative assay e.g. NGS or pyrosequencing Database interrogation ( Familial segregation studies (matrilineal) Tissue segregation studies (higher in post-mitotic tissue e.g. SKM; lower in mitotic tissue e.g. blood) McFarland et al Trends Genet., 20(12), Single muscle fibre segregation studies

10 % enzyme activity Our pipeline case study Patient presented with myopathic phenotype A B * * * Control Patient 0 Complex I Complex II Complex III Complex IV C

11 1. Database interrogation Lines of evidence 2. Predicted effects on mt-trna Pro structure and conservation analysis Aminoacyl acceptor stem Patient DHU Loop TΨC Loop m.15998a>t Anticodon Loop

12 3. Segregation patterns Lines of evidence 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 ± 0.62 (n=15) ± 6.09 (n=12) p< Hardy et al Neurol Genet, 2(4), e82

13 Homoplasmic mtdna variants mt-trna Val Cardiac SKM m.1624c>t McFarland et al Nat genet, 30,

14 Pathogenic homoplasmic mtdna variants 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 C Pat Pat C mt-trna Leu(UUR) mt-trna Cys Ile m.4300a>g Isolated hypertrophic cardiomyopathy Glu m.14709t>c Myopathy with diabetes

15 Discordance with functional studies Patient presented with NARP phenotype (strong prior likelihood of MTATP6/8 pathogenic variant) Blood n.d. MTATP6 m.8839g>c p.(ala105pro) detected SKM 58% Urine 76% Blood 26% Blood n.d Urine n.d. Blood n.d Urine n.d. BUT no evidence of complex V deficiency

16 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

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