The in cis T251I and P587L POLG1 base changes: Description of a new family and literature review

The in cis T251I and P587L POLG1 base changes: Description of a new family and literature review

Available online at www.sciencedirect.com ScienceDirect Neuromuscular Disorders 25 (2015) 333–339 www.elsevier.com/locate/nmd The in cis T251I and P...

698KB Sizes 33 Downloads 238 Views

Available online at www.sciencedirect.com

ScienceDirect Neuromuscular Disorders 25 (2015) 333–339 www.elsevier.com/locate/nmd

The in cis T251I and P587L POLG1 base changes: Description of a new family and literature review Carmela Scuderi a,*, Eugenia Borgione a, Filippa Castello a, Mariangela Lo Giudice a, Sandro Santa Paola a, Mariaconcetta Giambirtone a, Francesco Domenico Di Blasi b, Maurizio Elia c, Carmelo Amato d, Santina Città b, Catalda Gagliano a, Giuliano Barbarino a, Girolamo Aurelio Vitello e, Sebastiano Antonino Musumeci e a

Unità Operativa di Malattie Neuromuscolari, IRCCS Oasi Maria SS, Troina, Italy b Unità Operativa di Psicologia, IRCCS Oasi Maria SS, Troina, Italy c Unità Operativa di Neurologia e Neurofisiopatologia Clinica e Strumentale, IRCCS Oasi Maria SS, Troina, Italy d Unità Operativa di Neuroradiologia, IRCCS Oasi Maria SS, Troina, Italy e Unità Operativa di Neurologia per il Ritardo Mentale, IRCCS Oasi Maria SS, Troina, Italy Received 4 November 2014; received in revised form 7 January 2015; accepted 14 January 2015

Abstract Mutations in the polymerase gamma-1 (POLG1) gene, encoding the catalytic subunit of the mtDNA-specific polymerase-γ, compromise the stability of mitochondrial DNA (mtDNA) and are responsible for numerous clinical presentations as autosomal dominant or recessive progressive external ophthalmoplegia (PEO), sensory ataxia, neuropathy, dysarthria and ophthalmoparesis (SANDO), spinocerebellar ataxia with epilepsy (SCAE) and Alpers syndrome. POLG1 mutations result in extremely heterogeneous phenotypes which often have overlapping clinical findings, making it difficult to categorize patients into syndromes, and genotype–phenotype correlations are still unclear. We describe a new family with a particular spectrum of clinical signs, that carried the c.752C>T mutation in exon 3 (T251I) and the c.1760C>T in exon 10 (P587L) in cis. These mutations were associated in the proband and in her brother with the new probably pathogenic mutation c.347C>A in exon 2 (P116Q). The proband presented a progressive cognitive impairment, mild myopathy, dilated cardiac right atrium and posterior white matter mild signal alteration, while her brother had migraine, mild myopathy, palpebral ptosis and posterior white matter mild signal alteration. Their mother and their sister carried the in cis T251I and the P587L mutations. The first presented neurosensorial hypoacusia, fatigue, heart block and a cerebral arteriovenous malformation nidus, while the latter had borderline intellectual functioning and signs of muscular involvement. Their father, with the P116Q mutation, had diabetes and myopathy. The complexity of the genotype–phenotype correlations associated with POLG1 mutations is reinforced in this work as evidenced by the presence of different clinic features in patients carrying the same mutations. © 2015 Elsevier B.V. All rights reserved. Keywords: Gene mutation; Mental retardation; Mitochondrial disease; Polymerase gamma

1. Introduction Disorders of nuclear–mitochondrial intergenomic cross talk are the most common form of mitochondrial disorders (MDs), especially with regard to mutations of the polymerase gamma-1 (POLG1) gene [1], that may account for about 25% of MDs [2].

* Corresponding author. Unità Operativa di Malattie Neuromuscolari, IRCCS Oasi Maria SS, V. Conte Ruggero 73, 94018 Troina (EN), Italy. Tel.: +39 0935 936111; fax: +39 0935 653327. E-mail address: [email protected] (C. Scuderi). http://dx.doi.org/10.1016/j.nmd.2015.01.004 0960-8966/© 2015 Elsevier B.V. All rights reserved.

The POLG1 gene on chromosome 15q25 encodes the α subunit of polymerase γ [3], a nuclear-encoded protein involved in replication and repair of mitochondrial DNA (mtDNA) in the eukaryotic cell [4]. POLG1 mutations have been found in patients with autosomal dominant or recessive progressive external ophthalmoplegia (PEO), often complicated by other clinical signs [5] and in ataxic and hepatocerebral syndromes [2]. However POLG1 mutations are associated with a wide spectrum of clinical symptoms and genotype–phenotype correlations are still unclear, as identical POLG1 mutations can give rise to distinct disease phenotypes, with a wide variation in

334

C. Scuderi et al. / Neuromuscular Disorders 25 (2015) 333–339

age of onset, electron transport activities and either recessive or dominant pattern of inheritance [6]. In this work we describe a family with an unusual association of clinical signs, variably distributed between the family members, that carried the c.752C>T mutation in exon 3 (T251I) and the c.1760C>T in exon 10 (P587L) in cis, associated, in two of the siblings, with the new probably pathogenic mutation c.347C>A in exon 2 (P116Q). We report in detail the clinical characteristics and do an extensive revision of literature data about the T251I+P587L mutations. Our work confirms the complexity of genotype–phenotype correlations of POLG1 mutations. 2. Case report We examined 6 members (I-4, II-9, II-10, III-1, III-2, III-3) of an Italian non-consanguineous family, in 5 of whom we identified POLG1 gene mutations. Extensive clinical examinations, that included neurological, ophthalmological and cardiological examinations, hearing tests, EEG, EMG, brain MRI or CT, were carried out. Informed written consent was obtained. The family tree is shown in Fig. 1. The proband (III-3), an 8 year-old girl, was born at term after an uneventful pregnancy. During delivery, a programmed caesarean section, the mother had sudden cardiac arrest. In the neonatal period the child was hypotonic and had a weak cry and later on delayed psychomotor development became a prominent feature. At age 3, neurological examination showed muscular hypotonia, joint laxity, absent deep tendon reflexes, broadbased gait, scapular winging, accentuation of lumbar lordosis and flat feet. Cardiological examination and behavioral audiometry were normal. EEG showed diffuse slow rhythms. Blood CK and lactic acid were normal. When she came back to our observation at age 8, a slight gait improvement was observed, but she had fatigue and echocardiogram showed dilated right atrium and ventricle tricuspid regurgitation. Brain MRI showed mild white matter signal alteration in the posterior periventricular regions. EMG examination showed motor unit potentials (MUPs) of low amplitude and clearly polyphasic morphology, mixed with MUPs of slightly increased amplitude suggesting a slow rearrangement of the motor unit.

Electroneurography was normal. The first neuropsychological investigations showed mild intellectual disability (ID), gradually switching on moderate ID, during the following longitudinal assessments. She also presented a poor expressive language, usually using unintelligible word-phrase sentences. The brother (III-1), 14 years old, was born at term by normal delivery and after an uneventful pregnancy. His psychomotor development was normal. He presented migraine by age 7 and complained of fatigue during exercise by age 12. On neurological examination he had mild diffuse muscle hypotonia and flat feet. At age 14 left eyelid ptosis was observed. Cardiological examination, EEG, audiometric examination, blood CK and lactic acid were normal. Brain MRI showed mild white matter hyperintensity in the posterior periventricular regions. EMG examination showed slight increase in percentage of polyphasic potentials. Electroneurography was normal. The neuropsychological assessment, at the age of 12 years old, showed a normal cognitive and adaptive functioning. The sister (III-2), 10 years old, was born at term by normal delivery and after an uneventful pregnancy. She complained of leg pain after physical activity. Neurological examination showed mild diffuse muscle hypotonia, hypotrophy, mild diffuse muscle weakness, joint laxity, scapular winging and increase of lumbar lordosis. Laboratory investigations revealed increased lactic acid (46.90 mg%; n.v. 4.55–19.80 mg%). Cardiological examination, EEG, ECG, audiometric examination and brain MRI were normal. EMG showed no evidence of peripheral neuromuscular injury. Muscular biopsy was not performed. The neuropsychological investigation showed a borderline intellectual functioning. Concerning the expressive language, she also presented a restricted vocabulary. The father (II-9) was a 42 year old man. By age 38 he complained fatigue. At the age of 40 he presented diabetes mellitus. Deep tendon reflexes were absent on neurological examination. Brain TC was normal. EMG examination showed MUPs of low amplitude and clearly polyphasic. Electroneurography was normal. The neuropsychological assessment showed a normal cognitive and adaptive functioning. The mother (II-10), 37 years old, presented deafness from childhood. At age 29, during her latter labor, she had sudden

Fig. 1. Pedigree of the studied family. Arrow indicates the proband. Filled symbols represent affected individuals, semifilled symbols indicate heterozygote carriers and slash marks, deceased individuals.

C. Scuderi et al. / Neuromuscular Disorders 25 (2015) 333–339

335

Fig. 2. (A) Patient III-3 – Axial T2-image showed mild white matter rarefaction in the posterior periventricular regions (arrows); (B) Patient III-1 – Axial T2-image demonstrated mild white matter hyperintensity in the posterior periventricular regions (arrows); (C) Patient II-10 – Coronal T2-image revealed AVM nidus in the left parasagittal rolandic region (arrows).

cardiac arrest. She complained of migraine and fatigue. On neurological examination she had mild diffuse muscle weakness. Blood CK and lactic acid were normal. Brain MRI revealed an arteriovenous malformation nidus (AVM) in the left parasagittal rolandic region (Fig. 2). Audiometric examination disclosed neurosensorial hypoacusia. EMG showed MUPs of low amplitude and clearly polyphasic morphology; recruitment was precociously rich, compared to the force explicated, suggesting primitive muscle impairment. Electroneurography was normal. The patient refused the muscle biopsy. The neuropsychological evaluation detected a normal intellectual and adaptive functioning, with specific weakness related to verbal intelligence area, probably due to the deafness. She also presented mild anxiety with specific phobias concerning indoor environment and crowd. The maternal grandmother (I-4), 58 years old, presented deafness from childhood, and diabetes from age 49. She complained of muscular cramps and fatigue. Neurological examination showed mild limb girdles muscle weakness. Audiometric examination disclosed neurosensorial hypoacusia. Blood CK and lactic acid were normal. Electromyography showed MUPs of low amplitude and clearly polyphasic morphology, suggesting a primitive muscular impairment. Electroneurography was normal. Brain MRI revealed a small vasculopathic alteration in the left frontal supraventricular region. The paternal grandfather died of prostate cancer, the paternal grandmother by the age of 40 had multiple strokes, a paternal uncle suffered sudden cardiac death by the age of 54, a maternal aunt (II-5) had prelingual deafness and the maternal grandfather (I-3), suffered from depression and paranoid traits. 3. Materials and methods 3.1. Morphologic and biochemical analysis Muscle specimens of cases III-3, III-1, II-9 and I-4 were taken from the right biceps and 9 µm serial cross-sections were

obtained for histochemical stains according to standard procedures. Immunohistochemical analysis was performed for patient I-4 using the following monoclonal antibodies: anti-mouse Dystrophin (VECTOR), anti-α-Sarcoglycan, antiβ-Sarcoglycan, anti-δ-Sarcoglycan, anti-γ-Sarcoglycan, anti-βDystoglycan, anti-Caveolin-3 (SANTA CRUZ Biotechnology), anti-laminin α2 chain (merosin) (5H2; CHEMICON), antilaminin 2 α2 chain (4H8-2; ALEXIS Biochemicals) and antiDysferlin (NOVACASTRA Laboratories). Respiratory chain complexes I–IV activities were measured in total muscle homogenates as previously described [7] and related to the mitochondrial marker citrate synthase. 3.2. Molecular genetic analysis After informed consent, genomic DNA of cases III-3, III-1, III-2, II-9, II-10, and I-4 was extracted from peripheral blood using standard protocols. PCR primers (provided by request) were designed by the software Vector NTI Advance (Informax, Frederick, MD, USA) to amplify all exons with flanking intronic sequences of POLG1, except exon 1, which is not translated (RefSeq NM_002693.1), POLG2 (RefSeq NM_007215.3), ANT1 (RefSeq NM_001151.3) and Twinkle (RefSeq NM_021830.4) genes. PCR products generated using Taq DNA polymerase (Roche, Indianapolis, IN) were purified with DyeEx 2.0 spin kit (Qiagen). Sequencing reactions were performed in both forward and reverse orientations using the BigDye Terminator Cycle Sequencing kit (version 1.1) and analyzed on an ABI310 automated DNA sequencer. Patient sequence data were aligned for comparison with corresponding wild-type sequence. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence NM_002693.1 and the corresponding methionine is the first aminoacid residue of the protein sequence. Total mitochondrial DNA isolated from muscle of cases III-3, III-1, II-9, and I-4 was tested for mtDNA deletions by

336

C. Scuderi et al. / Neuromuscular Disorders 25 (2015) 333–339

long PCR and screened for mtDNA point mutations by amplifying the entire mtDNA in 24 partially overlapping PCR fragments as described elsewhere [8], and sequenced using BigDye Terminator Kit and an ABI 310 Genetic Analyzer. The measure of mtDNA copy number was performed on muscle biopsy by Quantitative Real Time PCR based on Taqman fluorescence. In this analysis, the amount of mtDNA was compared with the amount of the nuclear gene cluster encoding the 18S rRNA on chromosome 21, contained in the same sample. The mtDNA/18S rRNA ratio obtained in the patient’s samples was expressed as a percentage of the mean value obtained in control samples, which represent the 100% value. 4. Results 4.1. Morphologic and biochemical analysis Muscle biopsy of case III-3, performed at age 3, showed variation in fiber size, mild lipid accumulation, some fibers with reinforcement of subsarcolemmal oxidative enzyme activity and some central nuclei. Muscle biopsy of case III-1 showed variation in fiber size and some central nuclei. Case II-9 disclosed variation in fiber size, central nuclei and rare esterase-positive fibers. Case I-4 revealed variation in fiber size, multiple central nuclei and some esterase-positive fibers. Immunohistochemical analysis showed a normal signal in the muscular fibers. Biochemical analysis showed normal activities of the respiratory chain complexes I–IV in all patients. 4.2. Molecular genetics The entire coding sequence of POLG1 gene was PCRamplified and directly sequenced disclosing the presence in case III-3 of three heterozygous variants: the c.752C>T (exon 3, p.T251I), c.1760 C>T (exon 10, p.P587L) in cis and a previously unreported C>A change at the nucleotide position 347 (c.347C>A) in exon 2, changing a proline to a glutamine at the amino acid position 116 (p.P116Q). This variant was absent in more than 200 unrelated control chromosomes and the effect of the P116Q mutation was predicted as possible damaging using the polyPhen software program [9]. The P116Q amino acid change is located in the N-terminal proof-reading domain and interspecies comparison demonstrated that P116 residue is highly conserved in vertebrates. We also found two previously unreported synonymous changes: c.948G>A (p.K316K) and c.2028G>A (p.A676A). No mutations were identified in POLG2, ANT1 and Twinkle genes. Long PCR excluded the presence of mtDNA deletions and sequencing of entire mitochondrial genome did not reveal any pathogenic mutations but showed several known polymorphisms (Table 1). These polymorphisms, identified by sequencing, were analyzed by the software HaploGrep (http://haplogrep.uibk.ac.at) that assigned haplogroup W3a1. The mtDNA copy number analysis showed normal mtDNA content in muscle when compared to tissue and age matched pooled controls.

Table 1 mtDNA polymorphisms identified in our patients by sequencing. Locus

Nucleotide change

Position

Aminoacid change

MT-Dloop MT-Dloop MT-Dloop MT-Dloop MT-Dloop MT-Dloop MT-Dloop MT-Dloop MT-RNR1 MT-RNR1 MT-RNR1 MT-RNR1 MT-RNR1 MT-RNR2 MT-ND1 MT-ND2 MT-ND2 MT-ND2 MT-COI MT-COII MT-ATP6 MT-ATP6 MT-ND4 MT-ND4 MT-ND4 MT-ND5 MT-ND5 MT-ND5 MT-Cytb MT-Cytb MT-Cytb MT-Cytb MT-TT MT-Dloop MT-Dloop MT-Dloop

73 189 194 195 204 207 263 310 709 750 1243 1406 1438 2706 3505 4769 5046 5460 7028 8251 8860 8994 11674 11719 11947 12414 12705 13263 14766 15326 15784 15884 15930 16223 16292 16519

A>G A>G C>T T>C T>C G>A A>G T>CTC G>A A>G T>C T>C A>G A>G A>G A>G G>A G>A C>T G>A A>G G>A C>T G>A A>G T>C C>T A>G C>T A>G T>C G>C G>A C>T C>T T>C

Noncoding Noncoding Noncoding Noncoding Noncoding Noncoding Noncoding Noncoding Noncoding Noncoding Noncoding Noncoding Noncoding Noncoding T-A Synonymous V-I Synonymous Synonymous Synonymous T-A Synonymous Synonymous Synonymous Synonymous Synonymous Synonymous Synonymous Synonymous T-A Synonymous A-P Noncoding Noncoding Noncoding Noncoding

Segregation analysis in the family showed that the c.752C>T and c.1760C>T substitutions were inherited from the mother and the c.347C>A substitution was inherited from the father. The brother (III-1) had the same genotype of the proband, while the sister (III-2) had the same genotype of the mother. The grandmother (I-4) showed a wild type sequence. All maternal relatives studied showed the same haplotype of the proband and were negative for mutations in POLG2, ANT1 and Twinkle genes. Also the father was negative for the latter genes, while he was assigned to the haplogroup H13a1c. 5. Discussion POLG1 gene mutations compromise mtDNA stability and may promote accumulation of mtDNA point mutations, multiple deletions and depletion [5]. Recessive mutations tend to cause mtDNA depletion, are usually present in childhood and cause Alpers syndrome or other phenotypes that include liver failure, while dominant mutations tend to cause adult onset multiple secondary deletions of mtDNA and determine PEO phenotype [2]. Unfortunately, muscle analysis may or may not show mosaic

C. Scuderi et al. / Neuromuscular Disorders 25 (2015) 333–339

337

Table 2 Clinical phenotypes and the genetic characteristics of the cases so far reported in literature. Allele1

Allele 2

P587L

Sex

Age at onset

Age years

PEO/ ptosis

M

47

61

+/

M

7

43

+/+

P587L P587L

R853W T251I

F M

49 4

53 4

+/+ +/

P587L+ P589L P587L+ T251I

W748S W748S

M F

17 45

50

+/+

P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I

M M M M M

48 54 0.5 4 0.5

58 57

+/

P587L+ T251I

M

10

P587L+ T251I P587L+ T251I P587L+ T251I

F F F

44 4 58

P587L

53

41

R232G G848S K1191R

F M F

0.5 0.5 1

P587L+ T251I

R853Q

F

0.2

P587L+ T251I

E1136K

P587L+ T251I

R232G

F

P587L+ T251I P587L+ T251I

R275X A467T

F F

68 51

+/ +/

P587L+ T251I P587L+ T251I P587L+ T251I

A467T G848S P648R

F F M

60 51 67

/+ /+

P587L+ T251I

H932Y

31

+/+

P587L+ T251I

H932Y

40

/+

P587L+ T251I

G848S

80

+/+

P587L+ T251I

K1191N

39

+/+

P587L+ T251I P587L+ T251I

N1157S G588D

9 6

46 45

M M F F M M F M

47 52 75

45 61 45 60 48 15 73

65 35 74 68 56 36 80

Muscle weakness, exercise intolerance Muscle weakness

Neuropathy

+

After birth

0.6/dead

Other signs

Reference

Gastrointestinal dysmotility

[5]

Hearing loss, hypogonadism

[5]

Acute disseminated encephalomyelitis, drowsiness, spasticity

+

Sensory defect Heart block Hypoacusia + Torticollis, feeding difficulty, esotropia, nystagmus, central hypotonia

+ +

+

Cataract Motor delay Blurred vision, diplopia, choking episodes, limb weakness

+ + +

Chronic bronchitis

Axonal sensorimotor polyneuropathy

+/ +/ +/ +/ +/+ +/+ +/ +/ +/ +/ +/ /+

Dysphagia

Proximal weakness Atrial hypertrophy + + SANDO

Psychic regression

[18] [25]

[21] [26] [15] [16] [10] [10] [27]

[23] +

Developmental delay Developmental delay 0.5/dead

Hepatic diseases

+

+ +

+ +

MNGIE

Muscle weakness

+

2 weeks

59

Unsteadiness

+/+ +/ +/ +/ +/ +/ +/ +/ +/

P587L+ T251I P587L+ T251I P587L+ T251I

M F

+ Myoclonic seizure

+/+ +/+

H932W R709X V1106I G848S A467T M603L V1106I L304R L304R R227W N864S G848S

71

Myopathy

Muscle weakness +/muscle pain

Mental deterioration

P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I

M

62 63 adult adult adult 48 60

Ataxia

+

Developmental delay

T251I+ P587L T251I+ P587L T251I+ P587L T251I+ P587L R309L G848S 2354Gins R227W R807P

56 70 74

Epilepsy

+

+/+ 0.7

P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I P587L+ T251I

F F M M M F

Cognition disorders

+ Alpers MCHS

Fatigue

MCHS

Hypotonia weakness

Hypomyelinating peripheral neuropathy

Hepatocerebral MDS

Cyclic vomiting, pancreatitis Microcephaly, failure to thrive, hearing loss Infantile hepatocerebral mtDNA depletion Frequent vomiting

MR

[23] [23] [28]

[14] [15] [15] [15] [13,29] [13,29] [13,29] [13,30] [16]

[16] [16] [13] [17] [14] [18] [15] [15] [15] [15] [19] [20] [21] [10] [10] [10] [22] [12]

[23] [23]

Exercise intolerance, limb girdle weakness +

Retinitis pigmentosa, diabetes

+ +

OPMD-like, pigmentary retinopathy, dysphagia, dysphonia, lung cancer Cataract, arrhythmia

+

CPK abnormalities

[11]

+

Ocular bulbar weakness, hypothyroidism Stroke/ischemic episodes, headache/migraine

[11]

Exercise intolerance Muscle weakness Muscle weakness Muscle weakness

Cataract

+

[23] [23] [24]

[11]

[11]

[11] [11]

Legend – MNGIE (mitochondrial neurogastrointestinal encephalomyopathy); SANDO (sensory ataxic neuropathy, dysarthria and ophthalmoparesis); MCHS (myocerebrohepatopathy spectrum disorders); MDS (mitochondrial DNA depletion syndrome); MR (mental retardation); OPMD-like (oculopharyngeal muscular dystrophy-like); CPK (creatinine phosphokinase).

338

C. Scuderi et al. / Neuromuscular Disorders 25 (2015) 333–339

pattern staining of cytochrome C oxidase, ragged red fibers (RRFs), decrease electron chain activity or mtDNA deletions or depletion [2,6], so the diagnosis of these disorders often becomes challenging. In this work, we describe a new family with POLG1 mutations and an interesting association of clinical signs. The proband had the c.752C>T mutation in exon 3 (T251I) and the c.1760C>T in exon 10 (P587L) in cis and the new c.347C>A mutation in exon 2 (P116Q). She presented a severe clinical picture, characterized by progressive cognitive impairment, mild myopathy, dilated cardiac right atrium and posterior white matter mild signal alteration. Her brother, even though carrying on the same POLG1 mutations, had a less severe phenotype characterized by migraine, mild myopathy, palpebral ptosis and posterior white matter mild signal alteration. Also her mother (II-10) and her sister (III-2), despite having both the T251I and the P587L in cis, presented different clinical phenotypes. Both showed signs of muscular involvement, but the mother had a normal intellectual functioning, while the sister had a borderline intellectual functioning. Furthermore, in the mother deafness is likely not related to the mutations in the POLG1 gene because it was also present in the maternal grandmother, negative for these mutations. Similarly, it is improbable that the arteriovenous malformation is related to the mitochondrial picture. The father, with the P116Q mutation, had diabetes and myopathy. Surprisingly, the maternal grandmother, despite the presence of neurosensorial deafness, diabetes and muscle weakness, did not show pathogenic POLG1, POLG2, ANT1, Twinkle or mitochondrial DNA mutations, that suggests a different condition that we could not identify. Our patients did not show biochemical respiratory chain defects or mtDNA rearrangements, nor RRFs in muscle biopsy, but that does not exclude pathogenicity of these mutations, as well as was already described in other cases of the literature, presumably because this tissue is only mildly affected [2]. The T251I + P587L mutations are among the more common pathogenic POLG1 mutations [10,11] and are present in 1% of normal Italian controls [12]. The c.347C>A had never been previously reported. We think it may have a pathogenic role, since it was not found in a group of 200 unrelated control chromosomes, suggesting that this was unlikely to be a rare polymorphism and mostly likely underlies the observed phenotype. Moreover, the role of the affected amino acid is highly conserved during evolution in several species while the PolyPhen analysis (http://genetics.bwh.harvard.edu/pph/) predicts the variant as possibly damaging. Generally, the T251I + P587L mutations are described associated to each other in cis both in homozygous [13–15] or in heterozygous condition [10–24]. Since only the P587L mutation was reported alone [5,18,21], this mutation has been proposed as the pathogenic allele [15]. The main clinical picture is represented by PEO, in many cases complicated by other clinical signs such as epilepsy [21], neuropathy, hypogonadism [5], ataxia, hypoacusia, dysphagia [16]. Two sisters with MNGIE phenotype, aged 15 years, were also described [19].

Among classical POLG1 phenotypes, there are infantile cases with Alpers–Huttenlocher syndrome and childhood myocerebrohepatopathy spectrum (MCHS) [10]. A 6-month-old female with hepatocerebral MD and SMA-like syndrome was described [12]. However many cases presented with atypical and sometimes complicated phenotypes. In Table 2 we report in detail the clinical phenotypes and the genetic characteristics of the cases so far reported in literature. These data show an extremely wide phenotype among affected patients. It is noteworthy that often a more severe clinical picture and an earlier age at onset are present, apparently as dominantly inherited in patients in whom the mutation in the second allele has not been identified [10,15,16,23,27]. As already suggested, these patients may have mutations in intronic regions or promoter, or deletions of the same gene, or mutations in other genes involved in the synthesis and maintenance of the mtDNA [31], which could be more deleterious than a mutation of the second allele of POLG1 gene. However, the timing of disease expression and severity of phenotype are not always explained by the type of associated mutation, in fact the association with the G848S mutation has been described both in an infant with Alpers syndrome with onset at 0.5 years [10] and in an elderly man of 75 years with SANDO [20], this being another enigma of POLG1 diseases [6]. Our index case had many polymorphisms of mtDNA. Del Bo et al. [32] presented evidence suggesting that mutations in the exonuclease domain of POLG1, as the T251I, which is responsible for the proofreading activity of the protein, result in a high frequency of randomly distributed rare mtDNA point mutations, while Wanrooij et al. [33] found an enhanced accumulation of point mutations in addition to deletions specifically in the control region. Since the grandmother, with no mutations in the POLG1 gene, had the same polymorphisms, these cannot be considered a consequence of mutations of this gene. However, it is not ruled out a share in the phenotype. In fact, it has been hypothesized that polymorphisms of the mtDNA can modify the expression of pathogenic mutations of mtDNA [34] and this may also be true for mutations in the POLG1 gene. So, the severity of the clinical picture of this girl could be partly explained by the convergence in the same subject of three mutations in the POLG1 gene transmitted from each parent, and of mtDNA polymorphisms transmitted through the maternal lineage. In conclusion, our work strengthens the evidence of the complexity of genotype–phenotype correlations of POLG1 gene, as evidenced by the presence of several clinic pictures in subjects carrying the same mutations, probably related to still unknown genetic or epigenetic factors concurrently involved in the phenotype. References [1] Hudson G, Chinnery PF. Mitochondrial DNA polymerase-gamma and human disease. Hum Mol Genet 2006;15:244–52. [2] Chinnery PF, Zeviani M. 155th ENMC workshop: polymerase gamma and disorders of mitochondrial DNA synthesis, 21–23 September 2007, Naarden, The Netherlands. Neuromuscul Disord 2008;18:259–67.

C. Scuderi et al. / Neuromuscular Disorders 25 (2015) 333–339 [3] Walker RL, Anziano P, Meltzer PS. A PAC containing the human mitochondrial DNA polymerase gamma gene (POLG) maps to chromosome 15q25. Genomics 1997;40:376–8. [4] Ropp PA, Copeland WC. Cloning and characterization of the human mitochondrial DNA polymerase, DNA polymerase gamma. Genomics 1996;36:449–58. [5] Filosto M, Mancuso M, Nishigaki Y, et al. Clinical and genetic heterogeneity in progressive external ophthalmoplegia due to mutations in polymerase gamma. Arch Neurol 2003;60:1279–84. [6] Saneto RP, Naviaux RK. Polymerase gamma disease through the ages. Dev Disabil Res Rev 2010;16:163–74. [7] DiMauro S, Servidei S, Zeviani M, et al. Cytochrome c oxidase deficiency in Leigh syndrome. Ann Neurol 1987;22:498–506. [8] Rieder MJ, Taylor SL, Tobe VO, Nickerson DA. Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome. Nucleic Acids Res 1998;26:967–73. [9] Ramensky V, Bork P, Sunyaev S. Human non-synonymous SNPs: server and survey. Nucleic Acids Res 2002;30:3894–900. [10] Wong LJ, Naviaux RK, Brunetti-Pierri N, et al. Molecular and clinical genetics of mitochondrial diseases due to POLG mutations. Hum Mutat 2008;29:150–72. [11] Tang S, Wang J, Lee NC, et al. Mitochondrial DNA polymerase gamma mutations: an ever expanding molecular and clinical spectrum. J Med Genet 2011;48:669–81. [12] Ferrari G, Lamantea E, Donati A, et al. Infantile hepatocerebral syndromes associated with mutations in the mitochondrial DNA polymerase-gammaA. Brain 2005;128:723–31. [13] Lamantea E, Zeviani M. Sequence analysis of familial PEO shows additional mutations associated with the 752C→T and 3527C→T changes in the POLG1 gene. Ann Neurol 2004;56:454–5. [14] Stewart JD, Tennant S, Powell H, et al. Novel POLG1 mutations associated with neuromuscular and liver phenotypes in adults and children. J Med Genet 2009;46:209–14. [15] Horvath R, Hudson G, Ferrari G, et al. Phenotypic spectrum associated with mutations of the mitochondrial polymerase gamma gene. Brain 2006;129:1674–84. [16] Di Fonzo A, Bordoni A, Crimi M, et al. POLG mutations in sporadic mitochondrial disorders with multiple mtDNA deletions. Hum Mutat 2003;22:498–9. [17] Kollberg G, Jansson M, Pérez-Bercoff A, et al. Low frequency of mtDNA point mutations in patients with PEO associated with POLG1 mutations. Eur J Hum Genet 2005;13:463–9. [18] González-Vioque E, Blázquez A, Fernández-Moreira D, et al. Association of novel POLG mutations and multiple mitochondrial DNA deletions with variable clinical phenotypes in a Spanish population. Arch Neurol 2006;63:107–11.

339

[19] Van Goethem G, Schwartz M, Löfgren A, Dermaut B, Van Broeckhoven C, Vissing J. Novel POLG mutations in progressive external ophthalmoplegia mimicking mitochondrial neurogastrointestinal encephalomyopathy. Eur J Hum Genet 2003;11:547–9. [20] Weiss MD, Saneto RP. Sensory ataxic neuropathy with dysarthria and ophthalmoparesis (SANDO) in late life due to compound heterozygous POLG mutations. Muscle Nerve 2010;41:882–5. [21] Ashley N, O’Rourke A, Smith C, et al. Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations. Hum Mol Genet 2008;17:2496–506. [22] Taanman JW, Rahman S, Pagnamenta AT, et al. Analysis of mutant DNA polymerase gamma in patients with mitochondrial DNA depletion. Hum Mutat 2009;30:248–54. [23] Blok MJ, van den Bosch BJ, Jongen E, et al. The unfolding clinical spectrum of POLG mutations. J Med Genet 2009;46:776–85. [24] Ferreira M, Evangelista T, Almeida LS, et al. Relative frequency of known causes of multiple mtDNA deletions: two novel POLG mutations. Neuromuscul Disord 2011;21:483–8. [25] Harris MO, Walsh LE, Hattab EM, Golomb MR. Is it ADEM, POLG, or both? Arch Neurol 2010;67:493–6. [26] Tzoulis C, Papingji M, Fiskestrand T, Røste LS, Bindoff LA. Mitochondrial DNA depletion in progressive external ophthalmoplegia caused by POLG1 mutations. Acta Neurol Scand Suppl 2009;189:38–41. [27] Burusnukul P, de los Reyes EC Phenotypic variations in 3 children with POLG1 mutations. J Child Neurol 2009;24:482–6. [28] Aitken H, Gorman G, McFarland R, Roberts M, Taylor RW, Turnbull DM. Clinical reasoning: Blurred vision and dancing feet: restless legs syndrome presenting in mitochondrial disease. Neurology 2009;72:86–90. [29] Lamantea E, Tiranti V, Bordoni A, et al. Mutations of mitochondrial DNA polymerase γA are a frequent cause of autosomal dominant or recessive progressive external ophthalmoplegia. Ann Neurol 2002;52:211–19. [30] Agostino A, Valletta L, Chinnery PF, et al. Mutations of ANT1, Twinkle, and POLG1 in sporadic progressive external ophthalmoplegia (PEO). Neurology 2003;60:1354–6. [31] Chan SS, Copeland WC. DNA polymerase gamma and mitochondrial disease: understanding the consequence of POLG mutations. Biochim Biophys Acta 2009;1787:312–19. [32] Del Bo R, Bordoni A, Sciacco M, et al. Remarkable infidelity of polymerase gammaA associated with mutations in POLG1 exonuclease domain. Neurology 2003;61:903–8. [33] Wanrooij S, Luoma P, van Goethem G, van Broeckhoven C, Suomalainen A, Spelbrink JN. Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA. Nucleic Acids Res 2004;32:3053–64. [34] Scuderi C, Borgione E, Musumeci S, et al. Severe encephalomyopathy in a patient with homoplasmic A5814G point mutation in mitochondrial tRNACys gene. Neuromuscul Disord 2007;17:258–61.