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Case Reports
. 2020 Feb;97(2):276-286.
doi: 10.1111/cge.13652. Epub 2019 Nov 14.

Identification of a novel heterozygous guanosine monophosphate reductase (GMPR) variant in a patient with a late-onset disorder of mitochondrial DNA maintenance

Ewen W Sommerville  1 , Ilaria Dalla Rosa  2 , Masha M Rosenberg  3 , Francesco Bruni  1   4 , Kyle Thompson  1 , Mariana Rocha  1 , Emma L Blakely  1 , Langping He  1 , Gavin Falkous  1 , Andrew M Schaefer  1 , Patrick Yu-Wai-Man  5   6   7 , Patrick F Chinnery  8 , Lizbeth Hedstrom  3   9 , Antonella Spinazzola  2   10 , Robert W Taylor  1 , Gráinne S Gorman  1
Affiliations
Case Reports

Identification of a novel heterozygous guanosine monophosphate reductase (GMPR) variant in a patient with a late-onset disorder of mitochondrial DNA maintenance

Ewen W Sommerville et al. Clin Genet. 2020 Feb.

Abstract

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a late-onset, Mendelian mitochondrial disorder characterised by paresis of the extraocular muscles, ptosis, and skeletal-muscle restricted multiple mitochondrial DNA (mtDNA) deletions. Although dominantly inherited, pathogenic variants in POLG, TWNK and RRM2B are among the most common genetic defects of adPEO, identification of novel candidate genes and the underlying pathomechanisms remains challenging. We report the clinical, genetic and molecular investigations of a patient who presented in the seventh decade of life with PEO. Oxidative histochemistry revealed cytochrome c oxidase-deficient fibres and occasional ragged red fibres showing subsarcolemmal mitochondrial accumulation in skeletal muscle, while molecular studies identified the presence of multiple mtDNA deletions. Negative candidate screening of known nuclear genes associated with PEO prompted diagnostic exome sequencing, leading to the prioritisation of a novel heterozygous c.547G>C variant in GMPR (NM_006877.3) encoding guanosine monophosphate reductase, a cytosolic enzyme required for maintaining the cellular balance of adenine and guanine nucleotides. We show that the novel c.547G>C variant causes aberrant splicing, decreased GMPR protein levels in patient skeletal muscle, proliferating and quiescent cells, and is associated with subtle changes in nucleotide homeostasis protein levels and evidence of disturbed mtDNA maintenance in skeletal muscle. Despite confirmation of GMPR deficiency, demonstrating marked defects of mtDNA replication or nucleotide homeostasis in patient cells proved challenging. Our study proposes that GMPR is the 19th locus for PEO and highlights the complexities of uncovering disease mechanisms in late-onset PEO phenotypes.

Keywords: GMPR; PEO; mitochondrial DNA maintenance; multiple mtDNA deletions; whole exome sequencing.

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Conflict of interest statement

Nothing to declare.

Figures

Figure 1
Figure 1
Clinical, histopathologic and molecular characterisation of a patient harbouring a novel heterozygous c.547G>C GMPR variant. A, Ophthalmological features of the patient with PEO harbouring a novel heterozygous GMPR variant, highlighting bilateral ptosis and frontalis muscle hyperactivity. B, A skeletal muscle biopsy from the patient was subjected to (a) COX, (b) SDH, (c) sequential COX‐SDH histochemical reactions and (d) haemotoxylin and eosin (H&E) staining. Scale bar represents 100 μM. C, 13‐kb long‐range PCR assay of skeletal muscle mtDNA demonstrating multiple mtDNA deletions in the patient (lane 4) compared with aged‐matched controls (lanes 1 and 2) and a patient with a single, large‐scale mtDNA deletion (lane 3). D, Quantitative single‐fibre real‐time PCR assay reveals that the majority of COX‐deficient fibres exhibit clonally expanded multiple mtDNA deletions involving the MTND4 gene. E, Mitochondrial respiratory chain expression profile showing NDUFB8 (complex I), COX‐I (complex IV) and porin levels in individual patient skeletal muscle fibres. Each dot represents an individual muscle fibre, colour coded according to mitochondrial mass (very low, blue; low, light blue; normal, light orange; high, orange; and very high, red). Black dashed lines represent the SD limits for the classification of fibres. Lines adjacent to the X‐ and Y‐axis represent the levels of NDUFB8 and COX‐I (beige, normal; light beige, intermediate (+); light blue, intermediate (−); and blue, negative]. F, Family pedigree and Sanger sequencing confirmation of the novel c.547G>C GMPR variant in the index case [Colour figure can be viewed at http://wileyonlinelibrary.com]
Figure 2
Figure 2
Genetic characterisation of the novel GMPR variant in skeletal muscle. A, Amplification of control and patient skeletal muscle‐derived cDNA across GMPR exons 3‐7 and sequencing chromatograms showing wild‐type PCR products from control and patient skeletal muscle. NT, No template. B, Steady‐state GMPR protein levels in control and patient skeletal muscle homogenate. SDHA was used as a loading control. C, Subfractionation of (a) HEK293T and (b) HeLa cells subjected to immunoblotting with a GMPR antibody and markers for each subfraction: GDH and EF‐Tu—mitochondrial matrix; AIF—mitochondrial inner membrane space; NDUFB8—mitochondrial inner membrane; eIF4E—cytosol. All subfractions were prepared from the same HeLa or HEK293T lysate [Colour figure can be viewed at http://wileyonlinelibrary.com]
Figure 3
Figure 3
Assessment of nucleotide homeostasis and mtDNA machinery markers in skeletal muscle and cytosolic and mitochondrial dNTP measurements in quiescent cells. A, Steady‐state proteins levels of nucleotide homeostasis and mtDNA maintenance markers in control and GMPR patient skeletal muscle homogenates. GAPDH, α‐tubulin and porin were used as loading controls. OXPHOS subunits SDHB (CII) and ATP5A (CV) were also used as markers to confirm protein loading. B, Cytosolic (left) and mitochondrial (right) dNTP levels in quiescent GMPR mutant and control fibroblasts. C, Relative mtDNA copy number in GMPR patient and control proliferating (P) and quiescent (Q) fibroblasts. Relative mtDNA copy number was expressed as fold change relative to one proliferating control [Colour figure can be viewed at http://wileyonlinelibrary.com]
Figure 4
Figure 4
Nucleoid and mitochondrial network morphology in skeletal muscle, mtDNA depletion‐repletion studies and guanosine supplementation in non‐dividing cells. A, Confocal images of patient and control muscle sections labelled with antibodies to the mitochondrial membrane protein TOM20 (red) and DNA (green). B, Non‐dividing patient and control cells were depleted of mtDNA using the intercalating agent ethidium bromide for 14 days. Ethidium bromide was then removed and mtDNA replenishment was followed for 14 days. Relative mtDNA copy number was expressed as fold change relative to one control. Student's t test was performed for statistical comparison between control and patient cell mtDNA copy number. C,D, Patient and control fibroblasts were put into quiescence by serum starvation for 14 days with or without guanosine supplementation. mtDNA copy number (C) and mtDNA deletions (D) were assessed at the end of the treatment. Increasing amounts of DNA (10, 20, 40 and 80 μg) were used as template for long‐range PCR [Colour figure can be viewed at http://wileyonlinelibrary.com]
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