mutations are emerging as one of the leading causes of both paediatric and adult-onset mitochondrial disease associated with disruption of mitochondrial DNA maintenance. RRM2B
mutations represent the third most common cause of Mendelian PEO and multiple mitochondrial DNA deletions in adults (13%), following mutations in POLG
(27%) and PEO1
(14%), based on data from both Oxford and Newcastle centres. It is clear there is significant clinical overlap between the multiple mitochondrial maintenance genes. In our cohort, ophthalmoparesis was universal, usually severe and often associated with ptosis (90% of patients). Other prominent myopathic features included proximal muscle weakness (52% of patients) and bulbar dysfunction (42% of patients). The latter manifested as dysarthria, dysphagia, dysphonia, facial weakness and neck weakness. Sensorineural hearing loss (36% of patients) and gastrointestinal disturbance (19% of patients), including irritable bowel syndrome-like symptoms and low body mass index, were also relatively common findings. This is perhaps unsurprising, given that they are prominent features in children with mitochondrial DNA depletion secondary to RRM2B
mutations. The presence of significant bulbar weakness, hearing loss and gastrointestinal symptoms should guide clinicians towards RRM2B
genetic analysis before POLG
, given that these key features are more common components of the clinical spectrum (bulbar dysfunction 42% versus 37% and 12%; sensorineural hearing loss 36% versus 11% and 9%; and gastrointestinal symptoms 19% versus unreported and 9% in RRM2B
-related PEO syndromes, respectively) (Horvath et al., 2006
; Fratter et al., 2011
Although cerebellar ataxia was present in 39% of cases, other central features of mitochondrial disease were seen less frequently, such as cognitive impairment (13% of patients) and encephalopathy/stroke-like events (10% of patients). Cardiac complications were rare (10%) and non-fatal. Disturbance in renal function was present in three patients, resulting from obstructive uropathy (two patients) and glomerulonephritis (one patient), with no reports of proximal renal tubulopathy, a common finding in children with RRM2B mutations and mitochondrial DNA depletion.
Two patients developed solid tumours (Patient 12 had oral carcinoma, and Patient 18 had breast carcinoma). RRM2B
is a gene not only involved in mitochondrial DNA replication but also plays a critical role in DNA damage repair. p53R2, a p53-inducible homologue of the R2 subunit of ribonucleotide reductase, has been evaluated in different cancer types and is known to play a critical role in DNA damage repair and cancer cell proliferation (Zhang et al., 2011
). These data may suggest abnormalities in DNA repair; however, it is not possible to conclude whether RRM2B
mutations are oncogenic, owing to small sample size and lack of mutational analysis of tumour tissue.
To further understand the functional consequence of the RRM2B
mutations identified, we mapped the positions of the mutated amino acids on the tertiary p53R2 structure (Smith et al., 2009
) (). Many of the missense mutations identified appear likely to affect the iron-binding properties of p53R2, and hence impair the catalytic capability of the functional heterotetramer (two p53R2 subunits and two R1 subunits). Gly195, Phe202 and Ile224 are located around the iron-binding pocket. Although the effect of Phe202Leu may be orchestrated through subtle hydrophobic contacts, the effect of amino acids Gly195 and Ile224 is more blatant. Positioned adjacent to amino acids that contribute to the iron coordination environment, substitutions at these locations (p.Gly195Arg and p.Ile224Ser) will influence their amino acid neighbours and alter the coordination of the iron atom(s). Previous molecular modelling has indicated that p.Arg41Gln prevents formation of a salt bridge that is important in conformational changes that control iron binding (Smith et al., 2009
; Pitceathly et al., 2011
). p.Arg41Trp is also predicted to prevent formation of this salt bridge. Arg211 forms a salt bridge to Glu85, which is thought to be important in stabilization of the di-iron form (Smith et al., 2009
; Pitceathly et al., 2011
), and therefore p.Arg211Lys may also destabilize the di-iron subunit.
Figure 5 Location of missense RRM2B mutations on crystal structure. An image of the p53R2 dimer structure is shown (Protein Data Bank code 3HF1). The locations of 11 of the 12 amino acids altered by missense mutations identified in this study are shown on the (more ...)
Thr144, Arg186, Thr218 and Gly273 are all located at the end of, or between in the case of Gly273, α-helices and appear to stabilize the orientation of the helices. Mutation of these four amino acids may reduce protein folding efficiency and is associated with autosomal-recessive disease in our cohort. The effect of the p.Asp70Asn mutation cannot readily be predicted, as Asp70 lies in a poorly understood region of the protein between two helices. p.Ala349Gly could not be modelled because the crystal structure does not include the C-terminal portion of the protein. However, Ala349 is located within a conserved heptapeptide (amino acids 345–351) required for interaction with the R1 subunit (Tyynismaa et al., 2009
), and loss of this heptapeptide has been proposed as the pathological basis of the exon 9 truncating mutations (Tyynismaa et al., 2009
There was a clear relationship between phenotypic severity and genotype in the patients studied. Individuals harbouring recessively inherited compound heterozygous RRM2B
mutations (Patients 19, 20, 21 and 22) presented at an earlier age (mean age of onset 7 years) with a more severe and multisystem disorder, whereas patients with single heterozygous mutations, inferring autosomal-dominant transmission, had a later average age of disease onset (46 years), as is seen with PEO1
mutations (Horvath et al., 2006
), and developed a predominantly myopathic phenotype consisting of PEO, ptosis, proximal muscle weakness and bulbar dysfunction, with exceptions noted (Patients 3 and 14). The distinction between recessively and dominantly inherited mutations was also evident on histochemical analysis of skeletal muscle tissue, in which COX-deficient fibres were much more widespread in patients with recessively inherited compound heterozygous mutations ().
Clinical syndromes caused by dysfunction of the nuclear maintenance genes can be broadly classified into two groups: (i) mutations that cause mitochondrial DNA depletion, which are at the most severe end of the phenotypic spectrum; and (ii) mutations that predispose to accumulation of multiple mitochondrial DNA deletions. The latter is further subdivided into: (a) recessively inherited disease, which presents during childhood with multisystem involvement; and (b) dominantly inherited disease, which is milder, typically develops in adulthood, and is often tissue-specific. A review of the literature suggests RRM2B
generally conforms to these basic principles. The most severe form of RRM2B
-related mitochondrial disease is associated with mitochondrial DNA depletion (Bourdon et al., 2007
; Bornstein et al., 2008
; Acham-Roschitz et al., 2009
; Kolberg et al., 2009
; Spinazzola et al., 2009
). Clinical presentation occurs in the first 6 months of life, with a multisystem disorder characterized by muscle hypotonia and weakness, seizures, gastrointestinal dysmotility, respiratory insufficiency, hearing loss, lactic acidosis, renal tubulopathy and early childhood mortality. The first reported adult-onset case was in a 30-year-old female with mitochondrial neurogastrointestinal encephalopathy who harboured compound heterozygous missense mutations in RRM2B
and mitochondrial DNA depletion (Shaibani et al., 2009
mutations were subsequently associated with multiple mitochondrial DNA deletions in two large unrelated families with autosomal-dominant PEO and an identical heterozygous nonsense mutation that caused truncation of the translated p53R2 protein (Tyynismaa et al., 2009
). Further reports have demonstrated RRM2B
mutations with Kearns–Sayre syndrome and sporadic/familial PEO (Fratter et al., 2011
; Pitceathly et al., 2011
There are, however, notable exceptions to these rules. First, RRM2B
-related mitochondrial DNA depletion can potentially cause a relatively mild clinical phenotype (Shaibani et al., 2009
); and second, identical RRM2B
mutations are associated with a varied phenotypic severity, depending on whether they exist in homozygous, compound heterozygous or heterozygous states. We attempt to explain the latter finding using molecular modelling of the RRM2B
missense mutations identified in this study, which suggests the variants can be broadly divided into two groups: mutations that severely impair ribonucleotide reductase activity and cause autosomal-dominant disease through a dominant-negative effect; and mutations associated with autosomal-recessive disease, which are predicted to result in moderately decreased catalytic activity or decreased levels of functional protein through reduced protein folding efficiency. This is likely to be a consequence of the heterotetrameric structure of ribonucleotide reductase, which predisposes the enzyme to both a dominant-negative effect (competitive binding and inactivation of the enzyme) or a gain-of-function effect (competitive binding with altered function of the enzyme), alongside the loss of enzymatic activity that occurs with recessively inherited disease. There is also evidence that impaired assembly of the multiprotein structures occurs with some mutations, and there may be a dosage effect, whereby wild-type p53R2 appears to partially compensate for the mutant allele, thus ameliorating the clinical phenotype when present in a heterozygous state (Pitceathly et al., 2011
). This phenomenon is demonstrated in the present study by the p.Ile224Ser, c.48G>A, p.Arg41Gln and p.Arg211Lys variants. These four variants are associated with relatively late-onset PEO (fourth to eighth decades) and multiple mitochondrial DNA deletions when present as the only heterozygous change, as in Patients 1, 1.1, 2, 3, 7 and 8. However, when homozygous or compound heterozygous, these variants are associated with much more severe multisystem disease, either with mitochondrial DNA depletion in the case of p.Ile224Ser (homozygote reported by Bornstein et al., 2008
) and c.48G>A (compound heterozygous with another pathogenic RRM2B
mutation, Oxford Molecular Genetics Laboratory, unpublished data), or with multiple mitochondrial DNA deletions in the case of p.Arg41Gln and p.Arg211Lys (compound heterozygous patient reported by Pitceathly et al.
, 2009, and Patient 21 in this study, respectively).
Our data support previous reports that RRM2B
mutations can present with a PEO-plus/Kearns–Sayre syndrome phenotype akin to single mitochondrial DNA deletion disorders (Pitceathly et al., 2011
). Patients 19, 20, 21 and 22 all developed symptoms before the age of 20 years, with PEO and other features suggestive of Kearns–Sayre syndrome, such as hearing loss, cerebellar ataxia and endocrine disturbance, although pigmentary retinopathy and heart block were absent. We would, therefore, recommend RRM2B
genetic analysis in patients with PEO-plus or Kearns–Sayre syndrome before a muscle biopsy is performed to exclude a single mitochondrial DNA deletion, if there is a Mendelian pattern of inheritance. RRM2B
analysis should also be considered in patients with mitochondrial neurogastrointestinal encephalopathy if blood/urinary deoxyuridine and thymidine levels are undetectable and thymidine phosphorylase activity is normal in white cells and platelets, based on the previous report of mitochondrial neurogastrointestinal encephalopathy secondary to compound heterozygous missense mutations in RRM2B
(Shaibani et al., 2009
), Case 20 reported here and because gastrointestinal symptoms were a prominent finding in our cohort of adult patients.
Finally, we report the novel c.48G>A RRM2B variant and provide evidence to support its pathogenicity through aberrant splicing, partial intron retention and premature termination of translation and hence predicted absence of any functional protein from this allele. We report two families (Patients 1, 1.1 and 2) where heterozygosity for c.48G>A is associated with adult-onset mitochondrial disease. Because all other RRM2B mutations reported to date in this disease group are either missense or exon 9 truncating, our data further expand the molecular heterogeneity of RRM2B-related adult mitochondrial disease.