A novel de novo dominant mutation in ISCU associated with mitochondrial myopathy

Abstract

Background: Hereditary myopathy with lactic acidosis and myopathy with deficiency of succinate dehydrogenase and aconitase are variants of a recessive disorder characterised by childhood-onset early fatigue, dyspnoea and palpitations on trivial exercise. The disease is non-progressive, but life-threatening episodes of widespread weakness, metabolic acidosis and rhabdomyolysis may occur. So far, this disease has been molecularly defined only in Swedish patients, all homozygous for a deep intronic splicing affecting mutation in ISCU encoding a scaffold protein for the assembly of iron-sulfur (Fe-S) clusters. A single Scandinavian family was identified with a different mutation, a missense change in compound heterozygosity with the common intronic mutation. The aim of the study was to identify the genetic defect in our proband.

Methods: A next-generation sequencing (NGS) approach was carried out on an Italian male who presented in childhood with ptosis, severe muscle weakness and exercise intolerance. His disease was slowly progressive, with partial recovery between episodes. Patient's specimens and yeast models were investigated.

Results: Histochemical and biochemical analyses on muscle biopsy showed multiple defects affecting mitochondrial respiratory chain complexes. We identified a single heterozygous mutation p.Gly96Val in ISCU, which was absent in DNA from his parents indicating a possible de novo dominant effect in the patient. Patient fibroblasts showed normal levels of ISCU protein and a few variably affected Fe-S cluster-dependent enzymes. Yeast studies confirmed both pathogenicity and dominance of the identified missense mutation.

Conclusion: We describe the first heterozygous dominant mutation in ISCU which results in a phenotype reminiscent of the recessive disease previously reported.

Keywords: De Novo Mutation; Fe-s Cluster; Iscu; Mitochondrial Myopathy.

Figure 1

Histochemical analysis of the patient’s muscle biopsy. (A) Gomori trichrome stain showing fibre size variability. (B) Strongly reduced histochemical activity of succinate dehydrogenase. Few fibres showed succinate dehydrogenase-positive staining (arrows). (C) Cytochrome c oxidase (COX) staining showing scattered fibres with severe reduction of histochemical COX activity (arrows). (D) Perls staining demonstrating punctuate accumulation of iron in the patient’s muscle fibres. Inset is a positive control (spleen) for the Prussian blue reaction. Bars correspond to 100 µm.

Figure 2

Identification and characterisation of an ISCU mutation. (A) Schematic representation of the ISCU cDNA (NM_213595.2) and ISCU protein with the nucleotide/amino acid change identified in this study. The functional IscU-like domain is in red; the mitochondrial targeting sequence (MTS) is in yellow. (B) Phylogenetic conservation of the amino acid residue (Gly96, in green) affected by the missense mutation identified in the patient. (C) Electropherograms of the genomic region (gDNA) and transcript (cDNA) harbouring the ISCU mutation, and pedigree. (D) Immunoblot analysis of ISCU^G96V mutant protein expression and subcellular localisation. Control and patient fibroblasts were harvested by trypsination, permeabilised by digitonin treatment, and separated into a cytosolic and a mitochondria-containing membrane fraction. Total cell lysates as well as cytosolic and crude mitochondrial fractions were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analysed for ISCU and ATP synthase F1β subunit (mitochondrial marker) steady-state protein levels (left panel). Chemiluminescence signals of ISCU and F1β in total lysate samples were quantified, and values obtained from patient fibroblasts were expressed relative to control cells (right panel). Error bars indicate the SDs (n=3). UTR, untranslated region.

Figure 3

Growth analysis of mutant yeast strains. (A) The strain isu1Δisu2Δ harbouring plasmid pFL39 with the wild-type ISU1 gene or the mutant allele isu1 ^G97V was analysed for growth on various media. Equal amounts of serial dilutions of cells from exponentially grown cultures were spotted onto yeast nitrogen base (YNB) medium plus 2% glucose, 2% lactate or 2% glycerol. The growth was scored after 3 days of incubation at 28°C. (B) The strains isu1Δisu2Δ/ISU1 and isu1Δisu2Δ/isu1 ^G97V were transformed with pFL38/ISU1 or with the empty vector. Equal amounts of serial dilutions of cells from exponentially grown cultures were analysed for growth on YNB medium plus 2% glucose or 2% lactate after 4 days of incubation at 28°C. (C) Cell yield was calculated by growing cells on liquid medium containing glucose or lactate and measuring the optical density at 600 nm after 72 hours of growth. Error bars indicate the SDs (n=3).

Figure 4

Measurement of enzyme activities and iron content in yeast. (A) Aconitase activity was measured in whole-cell extracts from cells grown exponentially at 28°C in yeast nitrogen base (YNB) medium plus 0.6% glucose. (B and C) Succinate dehydrogenase activity and cytocrome c oxidase activities were measured in a mitochondria-enriched fraction obtained from cells grown as described before. The values for isu1Δisu2Δ/isu1 ^G97V and isu1Δisu2Δ/ISU1/isu1 ^G97V strains are expressed as percentage of the activities obtained in the strains isu1Δisu2Δ/ISU1 and isu1Δisu2Δ/ISU1/ISU1. (D) Cellular iron content was quantified in cells grown up to early stationary phase in YNB 0.2% glucose and 2% galactose medium. *<0.05 (unpaired two-tailed t-test), **<0.01 (unpaired two-tailed t-test). (E) Gal-ISU1/isu2Δ cells and isu1Δ cells expressing Myc-tagged Isu1 were radiolabelled with ⁵⁵Fe and ⁵⁵Fe incorporation into Isu1-Myc was determined by immunoprecipitation with α-Myc antibodies followed by scintillation counting. Wild-type cells harbouring the empty vector (e.v.) served as control. Isu1-myc protein levels in isu1Δ cells were determined by immunostaining with α-Myc antibodies. Porin (Por1) served as a loading control. (F) Gal-ISU1/isu2Δ cells expressing Isu1 from vector pFL39 and the reporter plasmid pFET3-GFP were cultivated in SD or SGal medium supplemented with 50 µM ferric ammonium citrate. At an optical density=0.5, the GFP-specific fluorescence emission of whole cells was determined. Error bars indicate the SDs (n=3).

Figure 5

In silico structural analysis. (A) Ribbon diagram of the Escherichia coli IscS (coloured in grey)-IscU (coloured in orange) complex (PDB ID: 3lvl). Close view of the Gly64 of IscU and Glu311, Ser312 and Met315 of IscS represented in Van der Waals and coloured by type. (B) Residues supposed to be involved in the co-ordination of the 2Fe-2S are represented as sticks.