A novel mutation in the tRNAIle gene (MTTI) affecting the variable loop in a patient with chronic progressive external ophthalmoplegia (CPEO)

Abstract

We describe a 62-year-old woman with chronic progressive external ophthalmoplegia (CPEO), multiple lipomas, diabetes mellitus, and a novel mitochondrial DNA (mtDNA) mutation at nucleotide 4302 (4302A>G) of the tRNA(Ile) gene (MTTI). This is the first mutation at position 44 in the variable loop (V loop) of any mitochondrial tRNA. The muscle biopsy revealed 10% ragged-red/ragged-blue fibers and 25% cytochrome c oxidase (COX)-deficient fibers. No deletions or duplications were detected by Southern blot analysis. The 4302A>G transition was present only in the patient's muscle and single-fiber analysis revealed significantly higher levels of the mutation in COX-deficient than in normal fibers. Like tRNA(Leu(UUR)), tRNA(Ile) appears to be a "hot spot" for mtDNA mutations causing CPEO.

Fig. 1

(A) Electropherogram of DNA sequence. Electrophoresed samples were analyzed with Sequence Analysis software (Perkin-Elmer Applied Biosystems). At position 4302 a heteroplasmic adenine to guanine transition is evident (4302A>G). (B) Nucleotide sequence showing inter-species similarity of the tRNA^Ile gene and the highly conserved base at nt 4302. The mutation in our patient is highlighted with a bold red letter and compared with human, bovine, mouse, Xenopus (X) laevis, Drosophila (D) yakuba. (C) PCR-RFLP analysis. Fragments after digestion with DdeI were separated on a 15% non-denaturing acrylamide gel, visualized and quantitated using SYBR Gold^® Nucleic Acid Gel Stain (Invitrogen). DdeI cuts the wild type into a 95 and a 78 bp fragment, whereas mutant mtDNA was digested into three (58, 37 and 78 bp). Patient’s tissues: M, muscle; B, blood; U, urine; C1, C2 (controls); uncut PCR product. (D) Secondary structure of the human wild type tRNA^Ile showing the nt. 4302 base pair substitution A-to-G at the variable loop (v loop) region. The red points show previously described mutations associated with CPEO phenotype (position 4267, 4274, 4285, 4298, and 4309).