The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

Infantile onset spinocerebellar ataxia (IOSCA) (MIM 271245) is a severe autosomal recessively inherited neurodegenerative disorder characterized by progressive atrophy of the cerebellum, brain stem and spinal cord and sensory axonal neuropathy. We report here the molecular background of this disease based on the positional cloning/candidate approach of the defective gene. Having established the linkage to chromosome 10q24, we restricted the critical DNA region using single nucleotide polymorphism-based haplotypes. After analyzing all positional candidate transcripts, we identified two point mutations in the gene C10orf2 encoding Twinkle, a mitochondrial deoxyribonucleic acid (mtDNA)-specific helicase, and a rarer splice variant Twinky, underlying IOSCA. The founder IOSCA mutation, homozygous in all but one of the patients, leads to a Y508C amino acid change in the polypeptides. One patient, heterozygous for Y508C, carries a silent coding region cytosine to thymine transition mutation in his paternal disease chromosome. This allele is expressed at a reduced level, causing the preponderance of messenger RNAs encoding Y508C polypeptides and thus leads to the IOSCA disease phenotype. Previously, we have shown that different mutations in this same gene cause autosomal dominant progressive external ophthalmoplegia (adPEO) with multiple mtDNA deletions (MIM 606075), a neuromuscular disorder sharing a spectrum of symptoms with IOSCA. IOSCA phenotype is the first recessive one due to Twinkle and Twinky mutations, the dominant PEO mutations affecting mtDNA maintenance, but in IOSCA, mtDNA stays intact. The severe neurological phenotype observed in IOSCA, a result of only a single amino acid substitution in Twinkle and Twinky, suggests that these proteins play a crucial role in the maintenance and/or function of specific affected neuronal subpopulations.

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We report here a novel human gene, hMRS3/4, encoding a putative mitochondrial transporter structurally and functionally homologous to the yeast mitochondrial RNA splicing proteins 3 and 4. These proteins belong to the family of mitochondrial carrier proteins (MCF) and are likely to function as solute carriers. hMRS3/4 spans V V10 kb of genomic DNA on chromosome 10q24 and consists of four exons that encode a 364-aa protein with six transmembrane domains. A putative splice variant, encoding a 177-aa protein with three transmembrane domains, was also identified. hMRS3/4 has a well-conserved signature sequence of MCF and is targeted into the mitochondria. When expressed in yeast, hMRS3/4 efficiently restores the mitochondrial functions in mrs3 o mrs4 o knock-out mutants. Ubiquitous expression in human tissues and a wellconserved structure and function suggest an important role for hMRS3/4 in human cells. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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