Introduction

Polymerase gamma (polγ or POLG), a holoenzyme, is the sole mitochondrial DNA polymerase enzyme encoded by a nuclear gene essential for synthesis, replication, and repair of mitochondrial DNA (mtDNA). Mutations in the POLG have emerged as one of the most common causes of inherited mitochondrial diseases in children and adults associated with either mtDNA multiple deletions or mtDNA depletions leading to mitochondrial dysfunction (Stumpf and Copeland 2011). Clinical presentation in patients with POLG mutations ranges from severe infantile to mild adult-onset manifestations, and it follows the autosomal dominant or recessive mode of inheritance (Blazquez-Bermejo et al. 2019; Stumpf and Copeland 2011; Tang et al. 2011). The major phenotypes associated with POLG mutations include Alpers–Huttenlocher syndrome, childhood myocerebrohepatopathy spectrum (MCHS), myoclonic epilepsy myopathy sensory ataxia (MEMSA), ataxia neuropathy spectrum (ANS), autosomal dominant progressive external ophthalmoplegia (adPEO), and autosomal recessive progressive external ophthalmoplegia (arPEO). The less frequently reported phenotypes include cardiomyopathy, palatal tremor, psychiatric illness, diabetes, distal myopathy, premature menopause, cataracts, Charcot-Marie-Tooth disease, idiopathic parkinsonism, and gastrointestinal pseudo-obstruction (Rahman and Copeland 2019; Tang et al. 2011).

Till date over 300 pathogenic mutations in the POLG have been reported (https://tools.niehs.nih.gov/polg) (Rahman and Copeland 2019). Of these, 176 unique POLG missense mutations associated with mitochondrial diseases are distributed uniformly in all domains of the protein (Nurminen et al. 2017; Piekutowska-Abramczuk et al. 2019). Although the prevalence of POLG mutations vary in different population groups, the most commonly reported POLG mutations are p.A467T, p.W748S, and p.G848S. There is no direct evidence of genotype-phenotype correlations, as the same mutations have been found in individuals with different phenotypes (Rahman and Copeland 2019). Knowledge regarding the POLG mutations in Indian population is inadequate. This study sought to define the genotype-phenotype spectrum of mitochondrial polymerase γ mutations in a large cohort of patients from a tertiary care hospital. Sequencing of all exons and intron-exon boundaries of POLG from 446 patients clinically diagnosed mitochondrial disorders with a wide spectrum of neuromuscular phenotypes revealed mutations in 19 patients (4.26%). Here, we describe the clinico-pathological and molecular spectrum of these 19 patients harbouring POLG mutations.

Patients and Methods

Clinical Data

The study was approved by the institute ethics committee, and informed consents were obtained from all the participants. This cohort was derived from the database of patients, who underwent a complete clinical and laboratory evaluation for mitochondrial etiology over a period of 9 years (2010–2018). Patients were recruited for the study when clinical, biochemical, histopathological, magnetic resonance imaging (MRI), nerve conduction studies, electroencephalography (EEG), and evoked potential studies suggested a probable diagnosis of mitochondrial disorder based on the criteria defined by Bernier et al. 2002. Standard definitions were used for the phenotypic characterization of POLG-related mitochondrial disorders (Bindu et al. 2015; Tang et al. 2011; Wong et al. 2008). A total of 446 cases with clinical diagnosis of mitochondrial disorders with neurological manifestations were analysed. The final cohort included 19 genetically confirmed patients.

Histological, Biochemical, and Long-Range PCR

Muscle biopsy was performed as part of investigative procedure with informed consent. Muscle histochemistry, respiratory chain enzyme activity, and ultrastructural studies were performed using standard protocols (Dubowitz 2013; Sonam et al. 2017; Govindaraj et al. 2011). Long-range PCR was used to check for the mitochondrial DNA deletions in muscle DNA (Hu et al. 2007).

Genetic Analysis of POLG Gene

Genomic DNA was extracted from blood and/or skeletal muscle tissue using standard protocol (Thangaraj et al. 2002). The entire coding regions, including intron–exon boundaries of POLG, were sequenced as described elsewhere (Van Goethem et al. 2001). The raw sequence data was analysed and edited using the Sequence Analysis and AutoAssembler tools. The edited sequences were aligned with POLG reference sequence (NM_002693), and variations noted were compared with Human DNA Polymerase Gamma Mutation Database (http://tools.niehs.nih.gov/polg/). Further, the functional impact of these variations in disease pathogenesis was predicted using in silico tools (Polyphen-2, Provean, and Mutation taster). The frequency of the identified variants was compared with 1000 genome database, Exome Aggregation Consortium (EXAC), dbSNP, and Genome Aggregation Database (gnomAD). In addition, we have compared the frequency of identified mutations in different populations.

Results

Clinical Characteristics of Patients with POLG Variations

A wide range of clinical features were observed in patients with POLG variations. The mean age at presentation was 19.8 years (age range 5–55 years, M: F = 10:9, children (n = 12, mean age = 10.3 years, age range = 1–18 years) and adults (n = 7, mean age = 36.2 years, age range = 25–55 years). Seven patients were born to consanguineous parents. Family history was positive in five (26%). Elevated serum lactate (23 to 68 mg/dL, ref range: 4.5–20 mg/dL) was observed in nine patients (47%). The most common neurological findings were ptosis (n = 10), peripheral neuropathy (n = 8), seizures (n = 5), and ataxia (n = 4). MRI brain done in 14/19 showed abnormality in eight. The abnormalities noted were mild cerebellar atrophy, basal ganglia signal changes, pallidal, and dentate nuclei mineralisation. Nerve conduction studies on thirteen patients showed length-dependent sensory motor axonal neuropathy in seven and both axonal and demyelinating neuropathy in three. The clinical phenotypes associated with POLG mutation (n = 19) in this study were chronic progressive external ophthalmoplegia (CPEO) and CPEO plus (n = 14), ataxia neuropathy (n = 3), encephalomyopathy (n = 1), and myopathy (n = 1). The associated clinical features of CPEO plus syndrome included proximal limb weakness, seizures, and peripheral neuropathy identified in five patients (Table 1).

Table 1 Clinical, histological, biochemical findings of patients with POLG variations
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Histological, Biochemical, and Genetic Findings

Skeletal muscle biopsy (n = 16) revealed characteristic features of ragged red fibres (RRF), ragged blue fibres (RBF), and cytochrome c oxidase (COX) deficient fibres in 14 patients, few rimmed vacuoles in one patient (P8) and significant myopathic features with mitochondrial changes in one patient (P17). Ultrastructural analysis done in two cases (P7, P9) who had p.L304R mutation showed mitochondrial abnormalities (sub-sarcolemmal mitochondrial aggregation, elongated and distorted mitochondria, paracrystalline inclusions). Mitochondrial respiratory chain analysis done in 14 patients showed isolated complex I deficiency in two patients, isolated complex IV in three, multiple complex deficiencies in six patients, and no deficiency in three. Long-range PCR revealed mtDNA multiple deletions in nine of 14 cases (Table 1).

POLG Variations

Analysis of exons and intron-exon boundaries of POLG from a cohort of 446 patients revealed six reported pathogenic mutations (p.H110Y, p.L304R, p.R627Q, p.W748S, p.R1148C, p.R1187W) and two novel variations (p.G132R and p.V1106A) in 19 patients (Fig. 1A). Of the 19 patients, twelve patients exhibit homozygous state, three had compound heterozygous, and four harboured heterozygous variation. Among eight mutations, three were located in exonuclease domain, two in the linker domain, and three in the polymerase domain. The most common pathogenic mutation was p.L304R, accounting for 42.10% (8/19), followed by p.W748S identified in four patients which existed in homozygous (P11 and P12) and compound heterozygous state (P13 and P14). The list of POLG variations is given in Table S1. The case vignettes of two patients with novel variations are described below.

Fig. 1
figure 1

a Domain structure of POLG and location of identified variants; reported variations are marked in black and novel in red. b Novel variation p.G132R in a 55-year-old woman with CPEO. a The pedigree information shows autosomal dominant pattern. Sequence electropherogram of c.394G>C shows (arrow) wild-type in control b, heterozygous in patient c, and brother d. Transversely cut skeletal muscle tissue shows ragged red fibre (× 200) e, COX deficient fibre (COX-SDH) (× 200) f, multiple mtDNA deletions g, and conservation of variation across different species h. c Novel variation p.V1106A in a 4-year-old girl with encephalomyopathy. a The pedigree information shows consanguinity. Sequence chromatogram shows (arrow) homozygous c.3317T>C in proband b and heterozygous carrier state in both parents c, d. Transversely cut skeletal muscle tissue shows ragged red fibre (× 200) e, COX deficient fibre (COX-SDH) (× 200) f, multiple mtDNA deletions g, and conservation of amino acid residues across different species h

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Novel Pathogenic POLG Variations

Patient No. 19: The heterozygous variant p.G132R was identified in a 55-year-old woman (P19; Table 1; Fig. 1B) who presented with history of slowly progressive bilateral ptosis and proximal muscle weakness of the lower limb of 10-year duration. There was no history of bulbar symptoms or upper limb weakness. There was no history of diplopia, diurnal variation, or fluctuation in the ptosis or muscle weakness. On examination, she had bilateral asymmetric ptosis left more than right, bilateral restricted extra ocular movements and bifacial weakness. Motor system examination revealed weakness of the neck muscles and proximal muscles of the lower limb. All deep tendon reflexes were sluggish to absent. Ophthalmological examination showed normal optic discs and retina. Neostigmine test was negative, and repetitive nerve stimulation test did not show any decremental response. Family history was positive, where proband’s father had ptosis and exercise intolerance and died at the age of 75 years. The patient’s 51-year-old brother developed mild ptosis at the age of 40 years. There was no weakness. Moreover, the variant was present in both patient and her brother with varying clinical symptoms. Histopathological study of skeletal muscle biopsy showed characteristic features of mitochondrial myopathy with 15% COX deficient fibres. Long-range PCR showed multiple mtDNA deletions in muscle DNA. Multiple alignment of POLG sequence revealed variation is highly conserved across the species and predicted to be deleterious by in silico tools. This variant was neither found in 200 healthy controls nor the 1000G and ExAC databases.

Patient No. 9: The homozygous variant p.V1106A was identified in a four-year-old girl (P9; Table 1; Fig. 1 C), who presented with delayed developmental milestones, particularly motor. She was noted to have drooping of eyelids, recurrent respiratory infections and vomiting from five months of age. At the age of 40 months, she developed status epilepticus following which there was global regression of attained milestones. Subsequently, she continued to have recurrent seizures of multiple semiologies including focal, myoclonic, and generalised tonic clonic seizures. Her younger brother had global developmental delay, ptosis, and hypotonia and died at 1 year of age. Examination showed failure to thrive and hypertrichosis, ptosis, eye movement abnormalities, normal ocular fundi, generalised hypotonia and hyporeflexia, choreoathetosis, and dystonia. Investigations revealed normal metabolic parameters and creatine kinase. Nerve conduction study showed sensorimotor axonal neuropathy. Histopathological study of skeletal muscle biopsy revealed RRF,RBF and COX deficient fibres (80%). Respiratory chain assays showed complex I deficiency (10% of the normal mean value). Multiple mtDNA deletions were observed. Segregation analysis revealed parents to be heterozygous carriers. This variation is highly conserved across species, absent in 200 control population samples, and predicted to be pathogenic by in silico analysis. The p.V1106A was neither found in 1000G nor ExAC.

Discussion

This study reports a large cohort of patients with varied phenotypes suggestive of POLG-related disorders in Indian population and is the first of its kind describing the molecular spectrum of polymerase γ mutations. We identified POLG variations in 4.26% (19/446) of patients consisting of eight different pathogenic mutations including two novel variations spanning across the functional domains of POLG protein. In our cohort, the patients with POLG mutations had varied age at presentation and spectrum of neurological manifestations. Seizures, developmental delay, ataxia, and peripheral neuropathy were noted predominantly. Several studies have shown the male preponderance in patient with POLG mutations (Ferrari et al. 2005; Horvath et al. 2006; Wong et al. 2008; Tang et al. 2011); however, there was no gender bias in our study. The most common phenotype observed was CPEO in both children and adults with age ranging from 5 to 55 years, similar to reported studies (Hanisch et al. 2015; Tang et al. 2011; Tzoulis et al. 2006). Among 14 CPEO patients, eight had peripheral neuropathy which implies the co-existence of neuropathy in CPEO that could be subclinical and may evolve over a period of time. It is therefore imperative to follow up these children with electrophysiological studies which aids in management. In addition, epilepsy was seen in patients carrying p.L304R mutation in our cohort. The most common seizure type noted was generalised tonic clonic seizures and status epilepticus.

Majority of the patients in our study had mitochondrial abnormalities as evidenced by abnormal histochemical findings (RRF and COX deficient fibres) and/or respiratory chain defect and genetic abnormalities (multiple mtDNA deletions) corroborating with the previous report (Tang et al. 2011). There was no correlation between muscle alterations and clinical severity. POLG mutations have also been reported in patients with normal muscle biochemistry (Blok et al. 2009). This indicates that the absence of mitochondrial alterations in muscle does not exclude the presence of POLG mutation. The p.L304R is the most common recessive pathogenic mutation accounting for ~ 21.05% of all mutant alleles observed in CPEO patients. The allelic frequency of the p.L304R was higher in our cohort compared with the previous study (Tang et al. 2011) (Table S2). In contrast, this mutation was present in low frequency in European populations (Ashley et al. 2008; Horvath et al. 2006; Rouzier et al. 2014). Our findings support that mutation p.L304R is predominant in South Asians (Tang et al. 2011). Further, this mutation was reported to be associated with various autosomal recessive phenotypes including CPEO, ataxia neuropathy, and Alpers syndrome (Tang et al. 2011). Other ethnic-specific POLG mutations reported are p.A467T, p.W748S, and p.T251I-p.P587I in European patients (Hakonen et al. 2005, 2007), p.R597W in Hispanic patients, and p.R1096C in Arabian patients (Tang et al. 2011).

The mutation p.W748S was the second most common in our study accounting for ~ 10.5% of all mutant alleles. The allelic frequency was lower (10.5%) compared with European populations (Blok et al. 2009; Horvath et al. 2006; Tang et al. 2011) (Table S2). In addition to p.W748S homozygous state, we also observed compound heterozygous state (p.W748S-p.E1143G) in two patients (P13 and P14) similar to the previous report (Hakonen et al. 2005). Functional evidence suggests the p.W748S exhibited low DNA polymerase activity, low processivity, and a severe DNA-binding defect. Further, in combination with p.E1143G can modulate the deleterious effect of the p.W748S mutation which has a detrimental effect on protein stability (Chan et al. 2006). The mutation pair p.W748S-p.E1143G is noted as the most common genetic cause of autosomal recessive ataxia in population of European descent (Hakonen et al. 2005; Hakonen et al. 2007). The phenotype associated with this variation in the present study included ataxia neuropathy, CPEO, and ataxia palatal tremor. The patient (P12) with palatal tremor is previously reported from our centre (Nagappa et al. 2015).

Homozygous mutation p.R627Q was identified in one CPEO patient (P10). This is a rare mutation found in cis/trans with other heterozygous pathogenic variants p.A467T, p.G11D, and p.Q1236H (Wong et al. 2008). A study has shown the p.R627Q to have DNA binding properties similar to wild type; however, the polymerase activity is reduced to 77% (Tang et al. 2011). Recently homozygous p.R627W has been reported to be associated with mitochondrial encephalopathy, seizures, and stroke-like episodes in consanguineous and non-consanguineous South Asian patients (Paramasivam et al. 2019). Two novel variants (p.G132R and p.V1106A) were identified in the present study. The heterozygous p.G132R noted in a CPEO patient (P19) and her brother had varying clinical features. This variant located in the polyglutamine stretch near the amino-terminal exonuclease domain is highly conserved but of unknown function. The patient had ptosis, muscle weakness, hearing loss, bilateral restricted extra ocular movements, and bifacial weakness, while her brother had only mild ptosis. The common clinical features were ptosis; hence, defects of other functionally related genes could be the possible cause for the severe clinical expression of the patient (P19). The other novel homozygous variant p.V1106A was in a patient with encephalomyopathy. This variant was previously reported in compound heterozygous state with p.W748S in a SANDO patient, and the effect of this variant in yeast model showed a mild defect of mtDNA stability (Kaliszewska et al. 2015). Another study described the variant p.V1106I (same position) as probable pathogenic in arPEO patient (Horvath et al. 2006).

In conclusion, we describe a large cohort of patients with POLG-related disorders. This study highlights the clinico-pathological and molecular spectrum in patients with POLG-related disorders from India and also reiterates the phenotypic heterogeneity seen among mitochondrial disorders. Extension of this study by employing the whole exome sequencing (WES)/whole genome sequencing (WGS) will unravel the genetic etiology in undiagnosed patients.