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. 2017 Mar 7;13(3):e1006620.
doi: 10.1371/journal.pgen.1006620. eCollection 2017 Mar.

Mutations in mitochondrial DNA causing tubulointerstitial kidney disease

Thomas M Connor  1 , Simon Hoer  2 , Andrew Mallett  3 , Daniel P Gale  4 , Aurora Gomez-Duran  5 , Viktor Posse  6 , Robin Antrobus  2 , Pablo Moreno  2 , Marco Sciacovelli  7 , Christian Frezza  7 , Jennifer Duff  8 , Neil S Sheerin  9 , John A Sayer  8 , Margaret Ashcroft  10 , Michael S Wiesener  11 , Gavin Hudson  8 , Claes M Gustafsson  6 , Patrick F Chinnery  5 , Patrick H Maxwell  12
Affiliations

Mutations in mitochondrial DNA causing tubulointerstitial kidney disease

Thomas M Connor et al. PLoS Genet. .

Abstract

Tubulointerstitial kidney disease is an important cause of progressive renal failure whose aetiology is incompletely understood. We analysed a large pedigree with maternally inherited tubulointerstitial kidney disease and identified a homoplasmic substitution in the control region of the mitochondrial genome (m.547A>T). While mutations in mtDNA coding sequence are a well recognised cause of disease affecting multiple organs, mutations in the control region have never been shown to cause disease. Strikingly, our patients did not have classical features of mitochondrial disease. Patient fibroblasts showed reduced levels of mitochondrial tRNAPhe, tRNALeu1 and reduced mitochondrial protein translation and respiration. Mitochondrial transfer demonstrated mitochondrial transmission of the defect and in vitro assays showed reduced activity of the heavy strand promoter. We also identified further kindreds with the same phenotype carrying a homoplasmic mutation in mitochondrial tRNAPhe (m.616T>C). Thus mutations in mitochondrial DNA can cause maternally inherited renal disease, likely mediated through reduced function of mitochondrial tRNAPhe.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Pedigree with maternally inherited renal disease and m.547A>T substitution.
(A) Pedigree of family showing individuals affected with chronic kidney disease (CKD, red symbols), and end-stage renal disease (ESRD, black symbols). One individual (yellow symbol) died many years previously age 18 with a diagnosis of Batten’s disease based on electronmicroscopy of a skin biopsy showing characteristic curvilinear bodies. We consider this to be unrelated to the renal disease (B) Renal biopsy showing evidence of focal tubular atrophy by light microscopy (arrowed). (C) Renal biopsy showing mitochondria in a renal tubular epithelial cell which appear structurally normal on electron microscopy. (D) Sanger sequencing of mtDNA from a control individual. (E) Sequence of an affected individual showing the homoplasmic m.547A>T substitution.
Fig 2
Fig 2. Mitochondrial function is impaired in m.547A>T fibroblasts and cybrids.
Oxygen consumption rate (OCR) was measured to assess mitochondrial function in fibroblasts (A, C) and cybrids (B, D), normalised for cell number. Representative comparison of fibroblasts (A) and cybrids (B) from control individuals (red) with fibroblasts or cybrids from patients with the m.547A>T substitution (blue) showing a substantial change in respiratory profile. There was a significant decrease in baseline oxygen consumption, and in maximal oxygen consumption following addition of FCCP in both fibroblasts (C) and cybrids (D). Asterisks *p < 0.05, **p < 0.005, and ***p < 0.001 for control versus patient groups, represented as the mean ± SD of separate experiments performed in triplicate with four patient and four control cell lines.
Fig 3
Fig 3. Reduced expression of mitochondrial heavy strand transcripts in m.547A>T patient-derived cells.
(A, C) RNA from patient- and control-derived cells (n = 4 each) was analysed for expression of a panel of mitochondrial tRNAs by quantitative Northern blot. The graphs show mean expression of heavy strand tRNAs relative to light strand tRNA Gln(Q). Values are expressed relative to the mean for the control cell lines in each case. Error bars indicate the standard deviation. Heavy strand tRNAPhe and tRNALeu expression is reduced relative to light strand tRNA Gln in m.547A>T fibroblasts (A, p<0.001) and cybrids (C, p<0.001 for tRNA Phe(F), Val (V) and Leu (L)). Experiments were repeated three times with equivalent results. Labelling for 5S rRNA was used to confirm equal loading. Heavy chain transcripts RNR1 (B) and CO1 (D) were measured by quantitative RT-PCR and were reduced in m.547A>T cybrids relative to the light strand transcript ND6. Each point represents the mean of several independent cybrid cell lines derived from a single donor.
Fig 4
Fig 4. m.547A>T fibroblasts display a marked reduction in complex I, III and IV protein expression.
Four individual pairs of fibroblasts from different patients and controls were SILAC labelled and the mitochondrial fraction was analysed by LC-MS/MS. The ratio of protein levels in patient versus control cells is displayed on a log2 scale. Respiratory chain proteins which were quantified in at least two pairs are shown. All identified mitochondrial-encoded proteins are annotated (ND1-5: NADH dehydrogenase subunit 1–5, CYT-B: cytochrome b, COII: cytochrome c oxidase II, ATP6: ATP synthase 6. See S4 Fig for complete annotation). The size of each dot indicates the significance (adjusted p value) of the difference in abundance of the protein in the patient and control samples. Location below the line of equivalence (0.0) indicates lower abundance in the patient samples compared to controls.
Fig 5
Fig 5. m.547A>T impairs mitochondrial heavy strand promoter activity.
(A) In vitro transcription with purified mitochondrial transcription factors and a dual promoter construct containing the heavy (HSP) and light strand promoter (LSP) was used to assess the effect of the m.547A>T substitution. The linear template results in run off (RO) transcripts of 190 nucleotides (nt) from the HSP and 90 nt from the LSP. (B) Metabolically labelled transcripts from wild type (wt) and m.547A>T variant promoter (A547T) were analysed by autoradiography. (C) Analysis of relative band intensities of (B) shows a significant reduction of HSP activity in the presence of the m.547A>T substitution (p = 0.0014). The experiment was performed three times yielding the same result.
Fig 6
Fig 6. mt.616T>C cybrids have a defect in respiration.
(A) Two pedigrees showing potential maternal inheritance of renal disease. Individuals with kidney disease are represented by filled shapes as in Fig 1. Family 8 from ref 13 forms part of pedigree II, and is indicated by the dotted box. Pedigree III is Family 6 from ref 13. Individuals from whom DNA was sequenced in the current study are marked with asterisks. All affected individuals who were sequenced were found to have homoplasmic levels of the m.616T>C substitution. The mitochondrial haplotype is T1a1. (B) Measurement of oxygen consumption in patient-derived and control cybrids showing a reduction in basal (before addition of oligomycin) and maximal respiration (after addition of FCCP) in patient-derived cybrids. (C) Expression levels of tRNAs quantified by Northern blot of three control and patient-derived cybrids show reduced mitochondrial tRNAPhe levels relative to the light strand encoded tRNAGln in mt.616T>C cells.(p = 0.006) The tRNA levels for valine and leucine were unaffected. (D) Conservation of mt tRNA Phe within vertebrates. The anticodon (GAA) is highlighted in bold, the nucleotides forming the last pair of the anticodon stem are underlined. Sequences were aligned with clustal omega and manually adjusted. The stem (s) and loop (l) secondary structure of the tRNA Phe of humans is indicated at the bottom.
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