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. 2009 Aug;85(2):290-5.
doi: 10.1016/j.ajhg.2009.07.009. Epub 2009 Aug 6.

A heterozygous truncating mutation in RRM2B causes autosomal-dominant progressive external ophthalmoplegia with multiple mtDNA deletions

Henna Tyynismaa  1 , Emil Ylikallio, Mehul Patel, Maria J Molnar, Ronald G Haller, Anu Suomalainen
Affiliations

A heterozygous truncating mutation in RRM2B causes autosomal-dominant progressive external ophthalmoplegia with multiple mtDNA deletions

Henna Tyynismaa et al. Am J Hum Genet. 2009 Aug.

Abstract

Autosomal-dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder that is characterized by accumulation of multiple mitochondrial DNA (mtDNA) deletions in postmitotic tissues. The disorder is heterogeneous, with five known nuclear disease genes that encode the proteins ANT1, Twinkle, POLG, POLG2, and OPA1. Defects in these proteins affect mtDNA maintenance, probably leading to stalled replication forks, consequent mtDNA deletion formation, and progressive respiratory chain deficiency. Here we present a large adPEO family with multiple mtDNA deletions, whose disease was not explained by mutations in any of the known adPEO loci. We mapped the disease locus in this family to chromosome 8q22.1-q23.3. The critical linkage region contained the RRM2B gene, which encodes the small subunit of the ribonucleotide reductase p53R2, which has previously been shown to be essential for the maintenance of mtDNA copy number. Mutation screening of RRM2B revealed a heterozygous nonsense mutation in exon 9 (c.979C-->T [p.R327X]) in all affected individuals that was absent in 380 control chromosomes. The same mutation was found to segregate in another adPEO family. The mutant mRNA escaped nonsense-mediated decay and resulted in a protein with truncation of 25 highly conserved C-terminal amino acids essential for the interaction with the ribonucleotide reductase subunit R1. We conclude that dominant-negative or gain-of-function mutations in RRM2B are a cause of multiple mtDNA deletions and adPEO.

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Figures

Figure 1
Figure 1
The adPEO Pedigrees The reduced pedigrees of the families show the participating members and their immediate relatives. The members of family 1 whose genotype data were used in the linkage analyses are marked with an asterisk (∗). The individuals marked with a question mark (?) were not clinically or genetically examined.
Figure 2
Figure 2
Analyses of Patient Muscle Biopsy for Mitochondrial Respiratory Chain Activity and mtDNA Quality and Quantity (A) Histochemical staining of frozen muscle section of a patient (family 1, IV:6) for cytochrome c oxidase (brown) and succinate dehydrogenase (blue) reveals abnormal mitochondrial respiratory chain activities, as indicated by arrows. Approximately 10% of the muscle fibers are affected. (B) Long-PCR analysis shows multiple mtDNA deletions in a muscle sample from an adPEO patient (family 1, IV:6). An 8.3 kb fragment of mtDNA was amplified. Similar multiple deletions are present in the muscle of adPEO patients that have a mutation in RRM2B, POLG, or C10ORF2, but not in a control sample. (C) mtDNA copy number is normal in muscle samples of adPEO patients (family 1, IV:6, IV:7, IV:8) as determined by quantitative real-time PCR as the ratio of the mitochondrial MT-CYB gene to the nuclear APP gene. Values are shown relative to the average of all control samples. The results were similar when the gene encoding mitochondrial 12S RNA was analyzed instead of MT-CYB. (D) mtDNA point mutation load is normal in a muscle sample of a patient (family 1, IV:6) as compared to the control range in both the control region of mtDNA and the MT-CYB gene.
Figure 3
Figure 3
Gene Sequence Analysis of RRM2B and Protein Analysis of p53R2 (A) Partial DNA sequence of normal RRM2B gene. (B) Partial DNA sequence of patient's RRM2B gene showing the heterozygous c.979C→T mutation. (C) RRM2B cDNA of patient (family 1, IV:7) myoblasts also contains the heterozygous c.979C→T mutation. We extracted total RNA from the cells with an RNeasy kit (QIAGEN). The RNA was used to generate cDNA with random primers and M-MLV Reverse Transcriptase (Promega) followed by amplification of part of the RRM2B cDNA with primers listed in Table S1 and Phusion polymerase (Finnzymes). (D) Western analysis of the p53R2 protein in myoblasts shows a truncated p53R2 protein present only in the patient sample (Pt) and not in the control samples (1–3). Ten micrograms of total cellular protein lysate was analyzed by western blot with p53R2 antibody (ab8105 rabbit polyclonal, Abcam). Actin was used as a loading control (sc-1616 goat polyclonal, Santa Cruz Biotechnology). (E) The C-terminal amino acids of the p53R2 protein are highly conserved. The R327 residue is indicated by an arrow, and the heptapeptide is underlined.
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