Using urine to diagnose large-scale mtDNA deletions in adult patients.

Kristin N Varhaug,Gonzalo S Nido,Per Knappskog,Pirjo Isohanni,Laurence A Bindoff,Irenaeus De Coo,Charalampos Tzoulis,Anu Suomalainen

doi:10.1002/acn3.51119

Abstract

ObjectiveThe aim of this study was to evaluate if urinary sediment cells offered a robust alternative to muscle biopsy for the diagnosis of single mtDNA deletions.MethodsEleven adult patients with progressive external ophthalmoplegia and a known single mtDNA deletion were investigated. Urinary sediment cells were used to isolate DNA, which was then subjected to long‐range polymerase chain reaction. Where available, the patient`s muscle DNA was studied in parallel. Breakpoint and thus deletion size were identified using both Sanger sequencing and next generation sequencing. The level of heteroplasmy was determined using quantitative polymerase chain reaction.ResultsWe identified the deletion in urine in 9 of 11 cases giving a sensitivity of 80%. Breakpoints and deletion size were readily detectable in DNA extracted from urine. Mean heteroplasmy level in urine was 38% ± 26 (range 8 ‐ 84%), and 57% ± 28 (range 12 – 94%) in muscle. While the heteroplasmy level in urinary sediment cells differed from that in muscle, we did find a statistically significant correlation between these two levels (R = 0.714, P = 0.031(Pearson correlation)).InterpretationOur findings suggest that urine can be used to screen patients suspected clinically of having a single mtDNA deletion. Based on our data, the use of urine could considerably reduce the need for muscle biopsy in this patient group.

Highlights

In humans, mitochondria are the only extra-nuclear organelles that have their own DNA; mitochondrial DNA
The first aim of this study was to evaluate if urinary sediment cells offered a robust alternative to muscle biopsy for the diagnosis of single mitochondrial DNA (mtDNA) deletions in adults
Our multicentre study shows that single deletions are detectable in urine in over 80% of the cases who had a clinical syndrome known to be caused by deletion, and in whom deletion was present in skeletal muscle

Summary

Introduction

Mitochondria are the only extra-nuclear organelles that have their own DNA; mitochondrial DNA (mtDNA). This 16.5 kb circular genome encodes 13 proteins that are subunits of respiratory chain complexes: the remaining protein subunits are encoded by genes within the nucleus. In addition to 13 proteins, the mtDNA encodes 22 tRNA and 2 rRNA that participate in mitochondrial translation. Multiple copies of mtDNA are present within each cell and a mutation in mitochondrial genome can affect some or all of the copies; coexistence of mutated and wild-type mtDNA is known as heteroplasmy. The first pathogenic mutations in mtDNA giving rise to human disease, were identified in 1988, and were single large-scale mitochondrial deletions (single deletions).[1].

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