Splice mutation in the iron-sulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance

Abstract

A myopathy with severe exercise intolerance and myoglobinuria has been described in patients from northern Sweden, with associated deficiencies of succinate dehydrogenase and aconitase in skeletal muscle. We identified the gene for the iron-sulfur cluster scaffold protein ISCU as a candidate within a region of shared homozygosity among patients with this disease. We found a single mutation in ISCU that likely strengthens a weak splice acceptor site, with consequent exon retention. A marked reduction of ISCU mRNA and mitochondrial ISCU protein in patient muscle was associated with a decrease in the iron regulatory protein IRP1 and intracellular iron overload in skeletal muscle, consistent with a muscle-specific alteration of iron homeostasis in this disease. ISCU interacts with the Friedreich ataxia gene product frataxin in iron-sulfur cluster biosynthesis. Our results therefore extend the range of known human diseases that are caused by defects in iron-sulfur cluster biogenesis.

Figure 1

Homozygosity Mapping by SNP Microarray in the Swedish Families Homozygosity mapping by SNP array in the three northern Swedish families with myopathy and deficiency of succinate dehydrogenase and aconitase. Each panel shows results from the affected individuals (patients P1 to P3) and the unaffected family member (individual H1). The horizontal band in each panel represents heterozygous signal from two-allele SNP markers distributed along chromosome 12. The gray shaded box indicates a region of homozygosity (loss of heterozygous signal) at 12q shared by all three affected individuals and not the unaffected son H1 of affected individual P3.

Figure 2

SNP-Array Genotyping and Mutation Detection in the Swedish Families Pedigrees and electropherograms of the ISCU mutation (g.7044 G→C) in the three families from northern Sweden, and SNP alleles flanking the ISCU gene. The positions of the SNP markers on chromosome 12 are indicated in Mb, with the corresponding alleles designated by A and B. AA or BB indicates that the proband is homozygous for SNP markers that surround the ISCU gene (the position is shown by an arrow). Double-headed arrows indicate a long stretch of homozygous markers. All patients share the same homozygous haplotype (highlighted in blue), consistent with a common origin.

Figure 3

Sequences of the Splice Mutation and the Additional Exon 4A The intronic Swedish myopathy mutation, and exon 4A. Genomic sequences of exons 4 and 5 with the corresponding protein residues indicated in purple. The intronic mutation in patients (g.7044 G→C), indicated by an arrow, extends a polypyrimidine tract (in green) and leads to the inclusion of an additional exon 4A (in blue) that is predicted to result in a premature stop codon (marked as ^∗) 53 bp upstream from the last exon junction. Numbers indicate ISCU protein-codon residues.

Figure 4

Abnormal ISCU Transcripts and Predicted Alteration of the Peptide Sequence (A) RT-PCR between exons 4 and 5, showing two different transcripts in patients and controls, the normal transcript containing exons 4 and 5, and one containing exons 4, 4A, and 5, as confirmed by cDNA sequencing. The retention of exon 4A is markedly enhanced in patient muscle. Both transcripts are present in the heterozygote. Note the presence of an additional larger transcript in patient muscle (gray arrowhead), for which cDNA sequencing revealed the presence of exons 4, 4A, and 5, as well as the retention of the intronic sequence between exon 4A and exon 5. These transcripts are in very low abundance, and their rare presence in control muscle as well (data not shown) strongly argues against their having a role in the disease. L indicates cDNA from lymphoblastoid cells from controls; P, H, and C indicate cDNA from muscle of the three northern Swedish patients, the heterozygote, and controls, respectively; M indicates 100 bp DNA ladder (Invitrogen); and ^∗ indicates no cDNA control. (B) Alignment of residues encoded by exons 4 and 5, showing the strong conservation of ISCU among species, as well as the alteration of the C terminus of ISCU resulting from the splice mutation in patients (SM). Numbers designate ISCU protein-codon residues, and the residues corresponding to the α6-helix are indicated.

Figure 5

Mitochondrial ISCU and Iron-Sulfur-Cluster-Containing Proteins in Skeletal Muscle Mitochondrial ISCU (m-ISCU) and iron-sulfur-cluster-containing proteins in muscle from northern Swedish patients (P1–P3), the unaffected offspring (H1), controls (C), and a patient with a myopathy due to a mitochondrial DNA deletion (M). (A) Western blots of m-ISCU and mitochondrial aconitase show a marked reduction in patient muscle. Note the increased m-ISCU level in the control patient with mitochondrial myopathy. (B) Aconitase enzymatic activity was determined after separation of cellular extracts in nondenaturing PAGE. The fast band corresponds to cytosolic aconitase (c-aconitase), and the slower band corresponds to the mitochondrial isoform (m-aconitase). The activities of both c- and m-aconitase are deficient in patients. Western blots of the iron-regulatory protein IRP1 also show reduced protein levels in patient muscle, consistent with the decrease in c-aconitase activity. (C) Activation of IRP1 to iron-responsive elements (IREs) with β-mercaptoethanol in skeletal muscles from northern Swedish patients (P1–P3), the unaffected offspring (H1), and controls (C). β-mercaptoethanol activates the binding of IRP1 to transcripts containing IRE by converting cytosolic aconitase to the IRE-binding form. There is no activation of IRE-binding in patients in contrast to controls, consistent with the decrease in aconitase activity in the patient samples.

Figure 6

SDH and Iron Staining in Skeletal Muscle Histochemistry of skeletal muscle from a northern Swedish patient (A and B), the heterozygote (C and D), and a control (E and F). Serial sections were stained for ferric iron (A, C, and E) and SDH (B, D, and F). Note the presence of intracellular iron overload in SDH-deficient fibers (marked with ^∗) in the patient sample. The punctate distribution of the iron staining is consistent with the mitochondrial iron overload previously detected in ultrastructural studies of these patients.