POLG1 p.R722H mutation associated with multiple mtDNA deletions and a neurological phenotype

<p>Abstract</p> <p>Background</p> <p>The c.2447G>A (p.R722H) mutation in the gene <it>POLG1 </it>of the catalytic subunit of human mitochondrial polymerase gamma has been previously found in a few occasions but its pathogenicity has remained uncertain. We set out to ascertain its contribution to neuromuscular disease.</p> <p>Methods</p> <p>Probands from two families with probable mitochondrial disease were examined clinically, muscle and buccal epithelial DNA were analyzed for mtDNA deletions, and the <it>POLG1, POLG2, ANT1 </it>and <it>Twinkle </it>genes were sequenced.</p> <p>Results</p> <p>An adult proband presented with progressive external ophthalmoplegia, sensorineural hearing impairment, diabetes mellitus, dysphagia, a limb myopathy and dementia. Brain MRI showed central and cortical atrophy, and ¹⁸F-deoxyglucose PET revealed reduced glucose uptake. Histochemical analysis of muscle disclosed ragged red fibers and cytochrome c oxidase-negative fibers. Electron microscopy showed subsarcolemmal aggregates of morphologically normal mitochondria. Multiple mtDNA deletions were found in the muscle, and sequencing of the <it>POLG1 </it>gene revealed a homozygous c.2447G>A (p.R722H) mutation. His two siblings were also homozygous with respect to the p.R722H mutation and presented with dementia and sensorineural hearing impairment. In another family the p.R722H mutation was found as compound heterozygosity with the common p.W748S mutation in two siblings with mental retardation, ptosis, epilepsy and psychiatric symptoms. The estimated carrier frequency of the p.R722H mutation was 1:135 in the Finnish population. No mutations in <it>POLG2</it>, <it>ANT1 </it>and <it>Twinkle </it>genes were found. Analysis of the POLG1 sequence by homology modeling supported the notion that the p.R722H mutation is pathogenic.</p> <p>Conclusions</p> <p>The recessive c.2447G>A (p.R722H) mutation in the linker region of the <it>POLG1 </it>gene is pathogenic for multiple mtDNA deletions in muscle and is associated with a late-onset neurological phenotype as a homozygous state. The onset of the disease can be earlier in compound heterozygotes.</p

Phylogenetic analysis of mitochondrial DNA:detection of mutations in patients with occipital stroke

Abstract A mitochondrial disorder may be one of the rare aetiologies of occipital stroke. Clinical and molecular analysis has suggested that 10% of young patients with occipital stroke have a mitochondrial disorder and 6% harbour the mutation 3243A>G in mitochondrial DNA (mtDNA), causing the MELAS syndrome. To identify other possible mtDNA mutations involved, we studied mtDNA genotypes in patients who had suffered an occipital stroke and in whom the common pathogenic mutations in mtDNA had been excluded. Since one systematic way of comparing mtDNA sequences is through phylogenetic analysis, a phylogenetic network for the Finnish mtDNA haplogroup U was constructed and used to identify differences in mtDNA between patients and controls. The usefulness of conformation sensitive gel electrophoresis (CSGE) for analysing differences within the coding sequence of mtDNA was also estimated. We studied mtDNA genotypes of 29 patients with occipital stroke. The aetiology of the stroke was assessed using the criteria of the Baltimore-Washington Cooperative Young Stroke Study, and migraine was diagnosed in 18 patients according to the International Headache Society criteria. Moreover, we studied the mtDNA genotypes of 42 patients with migraine and a total of 480 population controls who reported that they themselves and their mothers were healthy with respect to common clinical manifestations of mtDNA disease. The mtDNA haplogroups were detected by restriction fragment analysis and the mtDNA structures of 14 patients with occipital stroke and 43 subjects belonging to haplogroup U were examined by CSGE. The data acquired by CSGE were then used to construct a phylogenetic network for the Finnish mtDNA haplogroup U. We found CSGE to be a highly sensitive and specific method for screening mutations and polymorphisms in mtDNA. The sequence data on the 43 subjects belonging to the mtDNA haplogroup U were used to construct a phylogenetic network, which was found to be an unambiguous tree with few homoplasies that pointed to several previously unidentified common polymorphisms. The major branch of the network was U5, which seemed to be quite specific to the Finns. Branches representing haplogroups U2, U4, U7 and K could also be detected. Restriction fragment analysis of the patients with occipital stroke revealed that all those with migraine as a probable aetiology belonged to the mtDNA haplogroup U, suggesting that this genotype confers a risk of occipital stroke. In addition to the five patients with migrainous stroke, we analyzed the complete mtDNA coding sequences of nine other patients with occipital stroke belonging to haplogroup U by CSGE. Analysis of the phylogenetic network revealed an association of migrainous stroke with mtDNA haplogroup U5. Furthermore, the distribution of the mtDNA genotypes in the patients with stroke differed from that found in the controls. Four patients harboured potentially pathogenic mutations. CSGE proved to be an effective method for use in mitochondrial genetics, enabling us to construct an unambiguous network for the Finnish haplogroup U. Similar phylogenetic networks are required for the purposes of both medical genetics and population genetics. Such networks were found to be helpful in deciding between a rare polymorphism and a pathogenic mutation in patients with occipital stroke. Likewise, they enabled more detailed comparisons to be made between and within populations and allowed more accurate phylogenetic relationships to be determined

Phylogenetic Network for European mtDNA

The sequence in the first hypervariable segment (HVS-I) of the control region has been used as a source of evolutionary information in most phylogenetic analyses of mtDNA. Population genetic inference would benefit from a better understanding of the variation in the mtDNA coding region, but, thus far, complete mtDNA sequences have been rare. We determined the nucleotide sequence in the coding region of mtDNA from 121 Finns, by conformation-sensitive gel electrophoresis and subsequent sequencing and by direct sequencing of the D loop. Furthermore, 71 sequences from our previous reports were included, so that the samples represented all the mtDNA haplogroups present in the Finnish population. We found a total of 297 variable sites in the coding region, which allowed the compilation of unambiguous phylogenetic networks. The D loop harbored 104 variable sites, and, in most cases, these could be localized within the coding-region networks, without discrepancies. Interestingly, many homoplasies were detected in the coding region. Nucleotide variation in the rRNA and tRNA genes was 6%, and that in the third nucleotide positions of structural genes amounted to 22% of that in the HVS-I. The complete networks enabled the relationships between the mtDNA haplogroups to be analyzed. Phylogenetic networks based on the entire coding-region sequence in mtDNA provide a rich source for further population genetic studies, and complete sequences make it easier to differentiate between disease-causing mutations and rare polymorphisms

Phylogenetic Network of the mtDNA Haplogroup U in Northern Finland Based on Sequence Analysis of the Complete Coding Region by Conformation-Sensitive Gel Electrophoresis

Mutations in mtDNA have accumulated sequentially, and maternal lineages have diverged to form population-specific genotypes. Classification of the genotypes has been made based on differences found in restriction fragment analysis of the coding region or in the sequence of the hypervariable segment I. Both methods have shortcomings, as the former may not detect all the important polymorphisms and the latter makes use of a segment containing hypervariable nucleotide positions. Here, we have used conformation-sensitive gel electrophoresis (CSGE) to detect polymorphisms within the coding region of mtDNA from 22 Finns belonging to haplogroup U. Sixty-three overlapping PCR fragments covering the entire coding region were analyzed by CSGE, and the fragments that differed in their migration pattern were sequenced. CSGE proved to be a sensitive and specific method for identifying mtDNA substitutions. The phylogenetic network of the 22 coding-region sequences constituted a perfect tree, free of homoplasy, and provided several previously unidentified common polymorphisms characterizing subgroups of U. After contrasting this data with that of hypervariable segment I, we concluded that position 16192 seems to be prone to recurrent mutations and that position 16270 has experienced a back mutation. Interestingly, all 22 samples were found to belong to subcluster U5, suggesting that this subcluster is more frequent in Finns than in other European populations. Complete sequence data of the mtDNA yield a more reliable phylogenetic network and a more accurate classification of the haplogroups than previous ones. In medical genetics, such networks may help to decide between a rare polymorphism and a pathogenic mutation; in population genetics, the networks may enable more detailed analyses of population history and mtDNA evolution