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We describe the mapping and identification of the gene for hereditary myopathy with lactic acidosis (HML). HML is characterized by low physical performance, resulting in physical exertion that causes early exhaustion, dyspnoea and palpitations. Using an autosomal recessive mode of inheritance, we mapped the trait to chromosome 12q23.3-24.11, with a maximum lod score of 5.26. The 1.6-Mb disease-critical region contained one obvious candidate gene-ISCU-specifying a protein involved in iron-sulphur cluster assembly. IscU is produced in two isoforms; one cytosolic and one mitochondrial, coded for by different splice variants of the ISCU gene. Mutational analysis of all exon and intron sequences as well as 1000 bp of the promoter of the ISCU gene revealed one intron mutation that was specific for the disease haplotype. The mutation is located in a region with homology to the interferon-stimulated response element (ISRE), but we could not see any effect of the mutation on expression levels in vitro or in vivo. We did, however, observe a drastic difference in the splicing pattern between patients and controls. In controls the mRNA was, as expected, mainly in the mitochondrial form, while in the patients a larger mRNA transcript was predominant. Sequencing of the product revealed that the mutation activates cryptic splice sites in intron 5 resulting in aberrant mRNA containing 100 bp of the intron. To conclude, our data strongly suggest that an intron mutation in the ISCU gene, leading to incorrectly spliced mRNA, is the cause of myopathy with lactic acidosis in this family.

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulphur cluster assembly gene (ISCU) leading to incorporation of intron sequence into the mRNA. This results in a deficiency of Fe-S cluster proteins, affecting the TCA cycle and the respiratory chain. The proteins involved in the Fe-S machinery are evolutionary conserved and shown to be fundamental in all organisms examined. ISCU is expressed at high levels in numerous tissues in mammals, including high metabolic tissues like the heart, suggesting that a drastic mutation in the ISCU gene would be damaging to all energy-demanding organs. In spite of this, the symptoms in patients with HML are restricted to skeletal muscle, and it has been proposed that splicing events may contribute to the muscle specificity. In this study we confirm that a striking difference in the splicing pattern of mutant ISCU exists between different tissues. The highest level of incorrectly spliced ISCU mRNA was found in skeletal muscle, while the normal splice form predominated in patient heart. The splicing differences were also reflected at a functional level, where loss of Fe-S cluster carrying enzymes and accumulation of iron were present in muscle, but absent in other tissues. We also show that complete loss of ISCU in mice results in early embryonic death. The mice data confirm a fundamental role for ISCU in mammals and further support tissue-specific splicing as the major mechanism limiting the phenotype to skeletal muscle in HML.

Hereditary myopathy with lactic acidosis (HML) is caused by an intron mutation in the iron-sulfur cluster assembly gene ISCU which leads to the activation of cryptic splice sites and the retention of part of intron 4. This incorrect splicing is more pronounced in muscle than in other tissues, resulting in a muscle-specific phenotype. In this study, we identified five nuclear factors that interact with the sequence harboring the mutation and analyzed their effect on the splicing of the ISCU gene. The identification revealed three splicing factors, SFRS14, RBM39 and PTBP1, and two additional RNA binding factors, matrin 3 (MATR3) and IGF2BP1. IGF2BP1 showed a preference for the mutant sequence, whereas the other factors showed similar affinity for both sequences. PTBP1 was found to repress the defective splicing of ISCU, resulting in a drastic loss of mutant transcripts. In contrast, IGF2BP1 and RBM39 shifted the splicing ratio toward the incorrect splice form.