DGUOK recessive mutations in patients with CPEO, mitochondrial myopathy, parkinsonism and mtDNA deletions

Sir, We read with great interest the study by Ronchi et al. (2012) reporting for the first time the identification by next generation sequencing (MitoExome) of recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase) in five adult patients with mitochondrial DNA (mtDNA) multiple deletions and various phenotypes including mitochondrial myopathy with or without chronic progressive external ophthalmoplegia (CPEO), liver failure leading to liver transplantation and late-onset recurrent rhabdomyolysis, and adult-onset lower motor neuron syndrome with mild cognitive impairment. These five patients represented 5.6% of the entire cohort of patients carrying multiple mtDNA deletions in muscle (n = 90) studied by the authors, and 9.8% of those genetically undiagnosed (n = 51). Previously, recessive DGUOK mutations were associated with the infantile hepatocerebral form of mtDNA depletion (Mandel et al., 2001), as a consequence of DGUOK role in catalysing the first reaction of the purine salvage pathway, which is crucial for mtDNA maintenance (Ahmed et al., 2015; Wang, 2016). We recently implemented a next generation sequencing-based (Illumina, TruSeq Custom Amplicon) diagnostic panel dedicated to mitochondrial phenotypes characterized by disordered mtDNA maintenance (depletion and accumulation of mtDNA multiple deletions) including 25 genes (complete list available upon request), including DGUOK. Similar to Ronchi et al. (2012) we also identified two probands from independent families with recessive DGUOK mutations representing 2.2% of the entire pool of undiagnosed patients with multiple mtDNA deletions screened (n = 89). Furthermore, these two DGUOK families constituted 4.2% of our historical cohort of patients with mtDNA maintenance disorders that reached a genetic diagnosis (n = 48). We briefly describe these two families and the phenotypes associated with the DGUOK mutations. Family 1 includes six siblings, two of whom are affected by a similar phenotype. Clinical and laboratory findings are summarized in Supplementary Table 1. The proband, a 63-year-old female, started complaining of progressive bilateral ptosis from 30 years of age, in absence of diplopia. She underwent bilateral blepharoplasty. From the age of 40 she experienced slowly progressive lower limbs weakness, with growing difficulties in standing from a chair and climbing stairs. In recent years she also started complaining of upper limb weakness. Neurological examination showed bilateral ptosis, ophthalmoparesis, mild weakness of cervical muscles and proximal limbs muscles, more evident at lower limbs, weak deep tendon reflexes at upper limbs and reduced vibration sense of lower limbs. The laboratory examinations revealed elevated serum creatine kinase (CK) and pathologically elevated lactic acid after standardized exercise (Montagna et al., 1995). Muscle biopsy showed numerous ragged-red and COX-negative fibres (Fig. 1A–C). Needle EMG was remarkable for myopathic changes, whereas nerve conduction and repetitive nerve stimulation tests were normal. Abdominal ultrasound imaging documented a slight hepatic steatosis. Muscle biopsy histology and molecular analysis of mtDNA. (A) Haematoxylin and eosin stain of Patient 1 from Family 1 shows some hypotrophic fibres (arrows) and a few basophilic fibres, most evident subsarcolemmally, which correspond to ragged-red fibres (asterisks). (B) COX/SDH double stain reveals numerous COX depleted fibres (asterisks). (C) Gomori modified trichrome stain demonstrates two ragged-red fibres (asterisks). (D) Haematoxylin and eosin stain of Patient 3 from Family 2 shows myopathic changes with some hypotrophic basophilic fibres (asterisk). (E) COX/SDH double stain reveals numerous COX depleted fibres (asterisks) with one ragged-blue fibre displaying massive increase of SDH stain (arrow). (F) Gomori modified trichrome stain shows some ragged-red fibres (asterisks). (G) MtDNA copy number assessment in skeletal muscle shows that both probands from Families 1 and 2 present a similar significant depletion of mtDNA, >50% in Patient 3 from Family 2. Asterisks indicate statistical significance (*P < 0.0001, ANOVA test). (H) Long range PCR shows a massive accumulation of mtDNA multiple deletions in Patient 3 from Family 2, whereas in Patient 1 from Family 1 the presence of multiple deletions is modest. wt = wild-type. (I) Droplet digital PCR quantitative assessment of mtDNA multiple deletions determines 7% of deleted molecules in Patient 1 from Family 1 and 75% in Patient 3 from Family 2, confirming that in this latter case there is a combination of mtDNA depletion and high levels of mtDNA deleted molecules. Data are expressed as box-plot. Asterisks indicate statistical significance (*P < 0.001, unpaired t-test). Sanger sequencing failed to detect mutations in the POLG, TK2, TWNK (TWINKLE) and SLC25A4 (ANT1) genes (not shown). The proband’s brother, a 60-year-old male, presented with a progressive bilateral ptosis from 35 years of age, without diplopia. In the past 4 years he has also complained of weakness in lower limbs and difficulties in whistling. Clinical and laboratory findings are summarized in Supplementary Table 1. Neurological examination showed bilateral symmetric ptosis, mild ophthalmoparesis, mild weakness of orbicularis oculi, orbicularis oris and cervical flexor muscles reduced vibration sense in the lower limbs, and weak Achilles’ reflexes. The laboratory examinations revealed elevated serum CK and pathological elevation of lactic acid after standardized exercise. Needle EMG and ENG were normal. This patient did not undergo muscle biopsy. Hepatic investigations were unremarkable. These two siblings from Family 1 were both found to be compound heterozygotes for the c.3G>A (p.M1I) and c.586C>T (p.P196S) missense mutations in the DGUOK gene. The proband of Family 2 is a 67-year-old female presenting with an 8-year history of progressive bilateral ptosis, and a 5-year history of upper and lower limb weakness. Her 54-year-old sister has also ptosis, but no further information is available. Clinical and laboratory findings are summarized in Supplementary Table 1. Her medical history was remarkable for depression, bilateral reduced visual acuity, retinal detachment and cataract in the left eye, hypertension, atrial fibrillation, osteoporosis, and a surgically removed pulmonary carcinoid. At 62 years of age the patient underwent muscle biopsy that showed numerous ragged-red fibres and 40% COX-negative/SDH-positive fibres with focal inflammatory infiltrates (Fig. 1D–F). Due to the inflammatory infiltrates she was treated with a 1-year course of glucocorticoids with subjective slight improvement of the weakness and decrease of serum CK from >13 000 U/l to 200–400 U/l, but she eventually worsened. Since the age of 64 the patient has complained of lower limb stiffness, followed by bradykinesia, which worsened over time. At age 67, neurological examination showed bilateral symmetric ptosis and ophthalmoparesis, head drop with a dystonic rightward tilt, hypomimia (Fig. 2A), diffuse cogwheel rigidity and bradykinesia, and moderate weakness of the proximal upper and lower limbs (Supplementary Video 1). She had no tremor and presented a tendency to retropulsion. Her UPDRS-III score was 71. Cognitive profile was normal. Neuro-ophthalmological examination revealed reduction of visual acuity (3/10 in the right eye and motu manu in left eye). The left eye was remarkable for optic atrophy, whereas the right eye showed mild optic atrophy and bone-spicule pigment deposits in the lower temporal retina (not shown). Visual evoked potentials were markedly delayed and small in amplitude in both eyes. Audiogram showed a mild, bilateral, high-tone sensorineural hearing loss. Clinical phenotype of Family 2. (A) Patient 3 from Family 2 at age 65 years. The picture shows bilateral ptosis, head drop with a dystonic rightward tilt, and hypomimia (Supplementary Video 1). (B) Top left: 3D stereotactic surface projections (3D-SSP) obtained using Syngo-MI-Neurology (Siemens Healthcare) reveals clear hypometabolism in the occipital cortex (blue area). Top right: T1-MPRAGE (mm3) shows no significant atrophy. Bottom: 18F-FDG PET (coronal, sagittal and axial views) shows bilateral hypometabolism of the occipital cortex. (C) The 123I-FP-CIT (DatScan) single photon emission tomography (SPET) reveals bilateral striatal reduced uptake of the tracer, more prominent posteriorly and on the right. Simultaneous PET/MRI (Biograph mMR, Siemens) acquired for 25 min (1 mm3 isotropic T1 magnetization-prepared rapid acquisition gradient echo sequence, axial 4 mm T2-TSE, diffusion weighted imaging, susceptibility weighted imaging sequences and a simultaneously acquired list mode PET) 1 h after the administration of 18F-FDG (200 MBq), demonstrated bilateral hypometabolism of the occipital cortex (prevalent on the right side) without significant atrophy at T1 images (Fig. 2B). Furthermore, a relative hypometabolism of basal ganglia was observed, especially in the putamen (Fig. 2B). The patient performed also a 123I-FP-CIT (DatScan, GE Healthcare) single photon emission tomography (SPET) that revealed a marked, bilateral reduction of basal ganglia uptake, prevalent posteriorly (putamen) and on the right side (Fig. 2C). The patient had normal cardiac function, but a hypoxic restrictive respiratory insufficiency requiring mechanical, not invasive ventilation. A short trial with levodopa was ineffective. This proband from Family 2 was compound heterozygote for the c.462T>A (p.N154K) and c.749T>C (p.L250S) missense mutations in the DGUOK gene. Concerning the DGUOK mutations found in Family 1, the same p.M1T missense substitution due to a different nucleotide change (c.2T>C) in exon 1 has been previously described in a patient affected by hepatocellular insufficiency and combined respiratory chain deficiency (Slama et al., 2005). This patient died at 3 months and was homozygous for the mutation affecting the ATG translation initiation codon. The authors postulated that the next ATG translation initiation codon possibly used was at nucleotide positions 52–54 of the cDNA sequence, which should generate a 17 amino acid shorter protein, affecting the mitochondrial targeting sequence (Slama et al., 2005). Furthermore, the same mutation (c.2T>C) and two additional ones including ours (c.1A>G, c.3G>A), all affecting the initial methionine, have been subsequently reported in paediatric patients with combined respiratory deficiency (Freisinger et al., 2006), suggesting that this codon is a mutational hotspot. The second mutation, the p.P196S missense mutation, has never been reported before and is found in only one allele from a European individual (1/246 254, gnomAD). Based on the in silico prediction tools (PolyPhen-2, SIFT, Provean), this amino acid substitution is predicted to have a deleterious effect on the protein. In Family 2, the amino acid substitution p.N154K is reported in the ExAC database with a frequency of 0.0001153 (14/121 410), never found in homozygosity and limited to European population, and is the same as in Ronchi et al. (2012). In their study p.N154K was found in compound heterozygosity with other DGUOK mutations in two unrelated patients affected by late-onset CPEO, and the DGUOK activity was shown to be severely defective, supporting the pathogenic role exerted by the p.N154K missense change. The other amino acid substitution observed in the Family 2 proband, p.L250S, has been previously reported in homozygosity in a paediatric patient born from consanguineous parents and affected by hepatocellular insufficiency (Slama et al., 2005). Furthermore, the same mutation has been described in compound heterozygosity in two siblings affected by liver failure and combined respiratory deficiency (Freisinger et al., 2006). In vitro assessment of the p.L250S mutation catalytic activity demonstrated a drastic reduction (Wang et al., 2005). In fact, this mutation affects a conserved residue localized in the active site (Johansson et al., 2001) and the non-conservative amino acid substitution possibly disrupts the local protein structure, interfering with catalysis (Wang et al., 2005). Overall, the compound heterozygous DGOUK recessive mutations found in our two families are highly probable to be pathogenic, thus impinging on the nucleoside salvage pathway and leading to pathologic mtDNA maintenance. We studied the two main outcomes of altered nucleoside metabolism, the accumulation of mtDNA deletions and the mtDNA copy number in the two available muscle biopsies. As shown in Fig. 1G–I, both probands displayed a partial mtDNA depletion combined with variable accumulation of deleted genomes. We first assessed the mtDNA copy number by a well-established qPCR-based protocol (Giordano et al., 2014), which documented in both probands a consistent and similar reduction of ∼50–60% compared to control samples (Fig. 1G). Considering that mtDNA long-range PCR is a first-level screening providing non-quantitative information and based on the evidence that both probands presented multiple mtDNA deletions (Fig. 1H), we quantified them by a novel approach based on the use of droplet digital PCR (ddPCR, Bio-Rad). This method is based on duplex amplification with specific probes in MT-ND4 (major arc) and MT-DLOOP (control region), adapted from published qPCR methods (Krishnan et al., 2007; Phillips et al., 2014). Our ddPCR absolute quantification revealed that there was a consistent accumulation of multiple mtDNA deletions in Patient 3 (∼75% of deleted mitogenomes) and a slight but significant accumulation in Patient 1 (∼7%) (Fig. 1I). From a clinical standpoint both patients presented with CPEO and myopathy, but their phenotype diverged dramatically in terms of severity and multisystemic involvement. The Family 1 patients had a prevalent myopathic phenotype, compatible with the limited accumulation of mtDNA multiple deletions but similar mtDNA depletion when compared with the Family 2 proband. A common feature to all patients was an increased creatine phosphokinase (CPK) that in Patient 3 reached very high levels on one occasion without evidence of rhabdomyolysis, which recurred in one of the patients from Ronchi et al. (2012). Interestingly, the Family 1 proband also displayed hepatic steatosis, possibly reminiscent of the hepatic involvement characterizing the hepatocerebral paediatric phenotype (Mandel et al., 2001). Differently from Family 1 patients, the Family 2 proband displayed also visual, auditory and CNS involvement. Remarkably, this patient had parkinsonian features documented by clinical examination and imaging, a phenotype recurrent also with other mtDNA maintenance disorders (DiMauro et al., 2013; Giannoccaro et at., 2017). Noticeably, this patient combined a marked accumulation of mtDNA multiple deletions with mtDNA depletion. Both molecular defects have been recently reported in dopaminergic neurons from substantia nigra of idiopathic Parkinson’s disease patients (Bender et al., 2006; Dölle et al., 2016; Grünewald et al., 2016). In conclusion, our study provides additional information on adult cases associated with DGUOK recessive mutations, expanding the range of phenotypic expression, and shows variable combinations of mtDNA maintenance defects, depletion and accumulation of multiple deletions. The reason behind these different outcomes in terms of mtDNA instability remains poorly understood. Finally, promising results obtained by nucleoside supplementation in patients with a cognate disease leading to nucleotide imbalance due to TK2 mutations (Garone et al., 2014) cast hope for a rapid transition to similar therapeutic approaches also in DGUOK patients, as we increase their diagnostic recognition. This study received support from “Programma di ricerca Regione-Università 2010-2012” (PRUa1RI-2012-008 to V.C.) and the Italian Ministry of Health (Ricerca Corrente 2016 to V.C.). Supplementary material is available at Brain online.