Introduction

Adenine nucleotide translocator 1 (ANT1; OMIM #103220), mitochondrial replicative helicase Twinkle (OMIM #606075) and polymerase gamma (POLG; OMIM #174763) genes affect mtDNA stability, and their mutations cause autosomal dominant progressive external ophthalmoplegia (adPEO).1, 2, 3 POLG mutations have also been found in autosomal recessive PEO (arPEO) and it is thought that mutations in this gene could explain 45% of sporadic or familial PEO cases with multiple mtDNA deletions.4 Additional clinical presentations comprising mtDNA deletions have been described recently. They include autosomal recessive sensory ataxic neuropathy with dysarthria and ophthalmoplegia (SANDO), which is associated with mutations in either POLG5 or TWINKLE6 genes, parkinsonism with premature menopause, mitochondrial recessive ataxia syndrome (MIRAS), and juvenile spino-cerebellar ataxia-epilepsy syndrome (SCAE), all associated with POLG mutations.7, 8 Furthermore, mutations in POLG have also been associated with mtDNA depletion in patients affected by Alpers syndrome or by early hepatocerebral failure.9, 10, 11 In order to improve the molecular diagnosis in such patients, we have developed a new dHPLC assay for the complete screening of ANT1, TWINKLE and POLG coding regions and exon–intron boundaries. We report here the results of this screening in a series of 15 patients, illustrating the heterogeneity of phenotypes associated with mtDNA instability. All selected patients carried either multiple mtDNA deletions in muscle or mtDNA depletion in liver (Table 1).

Table 1 Clinical features of patients

Materials and methods

Subjects

The clinical findings and family histories are summarized in Table 1. The pedigrees are given in Figure 1.

Figure 1
figure 1

Pedigrees of six patients with POLG or TWINKLE mutations. +: presence of POLG mutation; −: absence of POLG mutation (families B, C, E, L, O); +: presence of TWINKLE mutation; −: absence of TWINKLE mutation (family G).

DNA extraction and PCR conditions

Blood samples were obtained from patients and all available family members after informed consent was given. Genomic DNA was extracted from leukocytes by standard procedure. POLG, ANT1 and TWINKLE genes were PCR amplified with primers listed in Supplementary Tables 1, 2 and 3. Different Taq DNA polymerases were used in order to obtain dHPLC best results for each amplified exon (Supplementary Table 4).

Cloning of wild-type DNA fragments

To detect homozygous variants, we generated wild-type DNA fragments. Each PCR product of ANT1, TWINKLE and POLG genes, resulting from control individuals, were subcloned into pGEM-T Easy Vector (Promega, Charbonnieres-Les-Bains, France). All wild-type clones were sequenced and then, used as controls in dHPLC.

dHPLC analysis

The dHPLC system used in this study is a Transgenomic Wave Nucleic Acid Fragment Analysis System (Transgenomic™, Crewe, UK). Optimal buffer gradients and mobile phase temperatures, which were determined by the Transgenomic software (Navigator™ software) are indicated in Supplementary Tables 1, 2 and 3. For homozygous variant detection, PCR products amplified from patients were combined with equal amounts of PCR products amplified from control DNA.

Sequencing analysis

PCR fragments were purified on Montage PCR columns (Millipore SA, SaintQuentin, France) and sequenced on a ABI 310 automated sequencer with D-Rhodamine cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA).

Results and discussion

Our principal aims were to determine the ANT1, TWINKLE and POLG mutational spectrum in a series of patients, and to develop an efficient molecular test for mutational analysis of genes involved in mtDNA stability. The entire ANT1, TWINKLE and POLG coding regions and exon–intron boundaries were screened by dHPLC in patients with neuromuscular symptoms. POLG only was analyzed in the three children deceased from hepatocerebral failure. All fragments giving an abnormal dHPLC profile were further analyzed by direct sequencing and a total of seven different probable pathogenic alleles were identified in six patients (Table 2A). Some of the dHPLC elution profiles are presented in Figure 2. Interestingly, the detection of A467T mutation in exon 7 of POLG was seen with one temperature only (Figure 2c). Several additional polymorphisms were also identified (Table 2B). In order to evaluate the reliability of our method, all fragments giving a normal dHPLC profile were also systematically sequenced and no discrepancy was observed between sequencing and dHPLC analysis.

Table 2 Mutations detected in the study
Figure 2
figure 2

dHPLC elution profiles of four POLG and one TWINKLE mutations. Patient status is indicated above elution profiles. DNA-heteroduplexes are indicated by an arrow. Amplicons and oven temperature are indicated. Hmz: Homozygous; Htz: Heterozygous. (a) Elution profiles of POLG exon 4 amplicons. From left to right: amplicon obtained from a control; amplicon from patient B, alone and mixed with a wild-type amplicon; amplicons from patient L and E, respectively. (b) Elution profiles of POLG exon 5 amplicons. Amplicon from a control (left) and patient C (right). (c) Elution profiles of POLG exon 7 amplicon. Amplicon from a control (left) and patient E (right) at 61.5°C (gray) and 62.8°C (black). (d) Elution profiles of TWINKLE exon 1. Amplicon from a control (left) and patient G (right) at 61.3°C (gray) and 61.8°C (black).

Table 3 Polymorphisms and variants detected in the study

Five patients out of 15 (33%) had POLG recessive mutations. POLG contains two domains, a DNA polymerase and a 3′–5′ exonuclease domain separated by a conserved inter-domain region known as spacer region.12, 13 Patient B, who presented with a SANDO phenotype, resulted to be homozygous for the c.911T>G mutation leading to a L304R amino-acid change (Table 2A). This highly conserved amino acid is located between exonuclease domains II and III of POLG. This mutation has already been reported in arPEO Belgian families presenting with features of the SANDO syndrome, but only in combination with the c.1399G>A substitution (A467T).5 We describe, for the first time, the L304R mutation in a homozygous state in a patient presenting with a SANDO phenotype.

The A467T mutation was found in a heterozygous state in patient C, in combination with a new c.1139G>A substitution. This new mutation leads to a G380A amino-acid change affecting a highly conserved residue lying between the exonuclease motifs ExoII and ExoIII. This patient, who presented with a PEO phenotype, was a sporadic case and the pathogenicity of this new mutation was impossible to be assessed by segregation analysis. Nevertheless, we did not find the c.1139G>A substitution (G380D) in 300 healthy french control chromosomes.The A467T substitution was also found in patient E, in combination with a new c.975_976insC mutation, which leads to a premature termination codon (T326fsX387). The A467T mutation, which maps within the conserved γ1 element of POLG spacer, has already been described in Alpers disease in a homozygous state or in combination with other mutations.10, 11 Another patient, who presented with an Alpers syndrome (patient O) also carried the A467T mutation in combination with a new c.1399G>A substitution, leading to a highly conserved T914P amino-acid change, between DNA polymerase domains A and B. The first mutation was inherited from the father, while the second was inherited from the mother. Recently, it has been shown that a single allele with the A467T substitution may manifest in a dominant manner, leading to late-onset ptosis.14 In our study, pedigree O analysis is consistent with this observation since the paternal grandfather, who is heterozygous for A467T, presented with muscular weakness but without PEO symptomatology (Figure 1). Finally, patient L was found to be a compound heterozygous carrying the c.911T>G (L304R) mutation in combination with the recently described c.2243G>C substitution (W748S). Our patient carried the W748S mutation in cis with a c.3428A>G (E1143G) variant. The L304R mutation was inherited from her mother while the W748S mutation, associated with E1143G, was inherited from her father. The W748S mutation is located in the conserved γ4 region of the POLG spacer, has been associated with SCAE, Alpers and infantile hepatocerebral syndromes,7, 9, 10, 15 and has always been described in combination with the E1143G variant in cis. From a practical point of view, the case of this young woman illustrates the interest of molecular analysis in mitochondrial diseases. She was pregnant and the identification of two POLG mutations in trans allowed us to give a reassuring genetic counselling.

One patient harbored a TWINKLE mutation which has already been described.1 Patient G was found to carry a c.1121G>A transition, predicting a highly conserved R374Q amino-acid change of the TWINKLE protein sequence. R374Q was not found in the patient’s daughter, the only healthy individual available in the pedigree. In our study, the R374Q mutation is associated with severe respiratory failure due to respiratory muscle weakness (pedigree G). No other TWINKLE or ANT1 mutations were found in the remaining pedigrees.

Overall, this study, which illustrates the variability of phenotypes associated with mtDNA stability defects, increases the mutational spectrum of POLG variants and provides an efficient and reliable detection protocol for ANT1, TWINKLE and POLG mutational screening.