Saara Finnilä

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.

Mitochondrial DNA polymorphisms as risk factors for Parkinson’s disease and Parkinson’s disease dementia

The activity of complex I of the mitochondrial respiratory chain has been found to be decreased in patients with Parkinson’s disease (PD), but no mutations have been identified in genes encoding complex I subunits. Recent studies have suggested that polymorphisms in mitochondrial DNA (mtDNA)-encoded complex I genes (MTND) modify susceptibility to PD. We hypothesize that the risk of PD is conveyed by the total number of nonsynonymous substitutions in the MTND genes in various mtDNA lineages rather than by single mutations. To test this possibility, we determined the number of nonsynonymous substitutions of the seven MTND genes from 183 Finns. The differences in the total number of nonsynonymous substitutions and the nonsynonymous to synonymous substitution rate ratio (Ka/Ks) of MTND genes between the European mtDNA haplogroup clusters (HV, JT, KU, IWX) were analysed by using a statistical approach. Patients with PD (n=238) underwent clinical examination together with mtDNA haplogroup analysis and the clinical features between patient groups defined by the number of nonsynonymous substitutions were compared. Our analysis revealed that the haplogroup clusters HV and KU had a lower average number of amino acid replacements and a lower Ka/Ks ratio in the MTND genes than clusters JT and IWX. Supercluster JTIWX with the highest number of amino acid replacements was more frequent among PD patients and even more frequent among patients with PD who developed dementia. Our results suggest that a relative excess of nonsynonymous mutations in MTND genes in supercluster JTWIX is associated with an increased risk of PD and the disease progression to dementia.

Evidence for mtDNA admixture between the Finns and the Saami.

Objectives: The Finns, and to a more extreme extent the Saami, are genetic outliers in Europe. Despite the close geographical contact between these populations, no major contribution of Saami mtDNA haplotypes to the Finnish population has been detected. Methods: To examine the extent of maternal gene flow from the Saami into Finnish populations, we determined the mtDNA variation in 403 persons living in four provinces in central and northern Finland. For all of these samples, we assessed the frequencies of mtDNA haplogroups and examined sequence variation in the hypervariable segment I (HVS-I). The resulting data were compared with published information for Saami populations. Results: The frequencies of the mtDNA haplogroups differed between the populations of the four provinces, suggesting a distinction between northern and central Finland. Analysis of molecular variance suggested that the Saami deviated less from the population of northern Finland than from that of central Finland. Five HVS-I haplotypes, including that harboring the Saami motif and the Asian-specific haplogroup Z, were shared between the Finns and the Saami and allowed comparisons between the populations. Their frequency was highest in the Saami and decreased towards central Finland. Conclusions: The high frequency of certain mtDNA haplotypes considered to be Saami specific in the Finnish population suggests a genetic admixture, which appears to be more pronounced in northern Finland. Furthermore, the presence of haplogroup Z in the Finns and the Saami indicates that traces of Asian mtDNA genotypes have survived in the contemporary populations.

Mitochondrial DNA haplogroup U as a risk factor for occipital stroke in migraine

exchange, followed by seven procedures with cryoprecipitate-poor plasma augmented with low-dose heparin (5–10 U/kg h). Neurological abnormalities resolved but there was no improvement in haematological indices (figure). Plasma exchange was discontinued after 18 days; the next day, fever and neurological disturbances recurred. Over the next 30 days, 18 staphylococcal protein A adsorption column (Prosorba) treatments and two doses of vincristine (1·5 mg/m) were given, and he had a splenectomy. His neurological function improved, but fever and haematological abnormalities persisted. 5 days after splenectomy his neurological condition worsened. Prosorba treatments were discontinued and he began a 10-day course of methylprednisolone at 40 mg/m twice daily. On the third day of steroids he was started on 13-cis retinoic acid (Accutane) 80 mg/m twice daily. Over the ensuing 16 days his platelet count steadily rose to normal (figure). He was discharged from hospital on accutane 80 mg/m twice daily for 6 months. He has now been off all medication for over 2 years without recurrence of TTP. Retinoids exert effects on cell differentiation and haemostatis. In-vitro studies have established that retinoids abrogate induction of procoagulant factors on endothelial cells and promote anticoagulant and fibrinolytic responses by induction of thrombomodulin and tissue plasminogen activator. In patients with acute promyelocytic leukaemia all-transretinoic acid effects resolution of the coagulopathy that accompanies that disorder. The many treatments this patient recieved preclude definitive attribution of his recovery to retinoic acid; nevertheless, there is a reason for further investigations of this new approach to treatment of TTP.

Phylogenetic analysis of mitochondrial DNA in patients with an occipital stroke: Evaluation of mutations by using sequence data on the entire coding region

Mitochondrial DNA (mtDNA) haplogroup U, defined by the polymorphism 12308A>G, may constitute a risk factor for an occipital stroke in migraine. We therefore identified 14 patients with an occipital stroke and with 12308A>G. We determined complete mtDNA coding region sequence for the patients and for population controls by conformation sensitive gel electrophoresis (CSGE) and direct sequencing. Sequence information was used to construct a phylogenetic network of mtDNA haplogroups U and K, which was found to be composed of subclusters U2, U4, U5 and a new subcluster U7, as well as cluster K. Five patients with a migrainous stroke belonged to subcluster U5 (P=0.006; Fishers exact test). Many unique mutations were found among the patients with an occipital stroke including two tRNA mutations that have previously been suggested to be pathogenic. Analysis of mtDNA sequences by CSGE and comparison of the sequences through phylogenetic analysis greatly enhances the identification of mtDNA clusters in population and detection of mtDNA mutations in patients.

Increase of collagen synthesis and deposition in the arachnoid and the dura following subarachnoid hemorrhage in the rat.

Arachnoidal fibrosis following subarachnoid hemorrhage (SAH) has been suggested to play a pathogenic role in the development of late post-hemorrhagic hydrocephalus in humans. The purpose of this study was to investigate the rate of collagen synthesis in the arachnoid and the dura in the rat under normal conditions and to study the time schedule and the localization of the increased collagen synthesis following an experimental SAH. We found that the activity of prolyl 4-hydroxylase, a key enzyme in collagen synthesis, was 3-fold higher in the dura than that in the arachnoid and was similar to the activity in the skin. We then induced SAH in rats by injecting autologous arterial blood into cisterna magna. After SAH, we observed an increase in prolyl 4-hydroxylase activity of the arachnoid and the dura at 1 week. At this time point the enzyme activity in both tissues was 1.7-1.8-fold compared to that in the controls and after this time point the activities declined but remained slightly elevated at least till week 4. The rate of collagen synthesis was measured in vitro by labeling the tissues with [(3)H]proline. The rate increased to be 1.7-fold at 1 to 2 weeks after the SAH in both of the tissues. Immunohistochemically we observed a deposition of type I collagen in the meninges at 3 weeks after the SAH. SAH is followed by a transient increase in the rate of collagen synthesis in the arachnoid and, surprisingly, also the dura. Increased synthesis also resulted in an accumulation of type I collagen in the meningeal tissue, suggesting that the meninges are a potential site for fibrosis. The time schedule of these biochemical and histological events suggest that meningeal fibrosis may be involved in the pathogenesis of late post-hemorrhagic hydrocephalus.

Adult‐onset ataxia and polyneuropathy caused by mitochondrial 8993T→C mutation

The 8993T→C mutation in mitochondrial DNA (mtDNA) has been described previously to be associated with infantile‐ or childhood‐onset phenotypes, ranging from Leighs syndrome to neurogenic weakness, ataxia, and retinitis pigmentosa syndrome. We report a kindred with adult‐onset slowly progressive ataxia and polyneuropathy and with the heteroplasmic 8993T→C mutation. Our findings suggest that the 8993T→C mtDNA mutation should be considered in the differential diagnosis of nondominant adult‐onset ataxia and axonal neuropathy. Ann Neurol 2005;58:337–340

Phylogenetic analysis of mtDNA haplogroup TJ in a Finnish population.

AbstractAn association between mitochondrial DNA (mtDNA) mutations 11778G>A and 14484T>C and mtDNA haplogroup J suggests that this haplogroup harbors substitutions capable of modifying the phenotype of Lebers disease. Our knowledge of the compilation of substitutions in haplogroup J is based on only a small number of complete mtDNA sequences, however. We constructed phylogenetic networks for mtDNA haplogroup TJ that were based on the sequence of the complete coding region and the hypervariable segment I, respectively, in 28 Finnish samples. The networks revealed a subdivision of the haplogroup into subclusters T1, T2, J1, and J2, while comparison of the two networks suggested nine fast evolving nucleotide sites in the hypervariable segment I. Genotypes of patients harboring 11778G>A or 14484T>C were obtained from the literature and were then placed in the network. Only four substitutions were found to be common to the patients, but none of these was unique to haplogroup J. If increased penetrance of the 11778G>A and 14484T>C mutations in patients belonging to haplogroup J is assumed, combinations of ancient substitutions must be implicated.

A novel mitochondrial DNA mutation and a mutation in the Notch3 gene in a patient with myopathy and CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterized by cerebral symptoms, but peripheral nerve or muscle involvement has not been reported. We describe a patient who had a stereotypic clinical presentation of CADASIL and, in addition, myopathy with ragged-red fibers, suggesting a mitochondrial disorder. Therefore we determined the nucleotide sequence in the entire coding region of the patients mtDNA by conformation-sensitive gel electrophoresis and sequencing. Sequence of the exon 4 in the Notch3 gene was determined in a similar fashion. We found that the patient had myopathy with ragged-red fibers, and ultrastructural examination revealed mitochondrial aberrations. CADASIL was due to an R133C mutation in Notch3; in addition, we found a novel mutation 5650G>A in the tRNAAla gene in mtDNA. The mutation was heteroplasmic, with the proportions of the mutant genome being 99% in muscle, 96% in the buccal epithelium, 95% in the skin, and 65% in the blood. The absence of the mutation in a maternal cousin four times removed indicated that it was new in the pedigree. We suggest that the mtDNA mutation is pathogenic, as it was associated with a relevant clinical phenotype, it was not found among controls, and it altered a structurally important segment in the amino acid acceptor stem in the tRNAAla. Furthermore, its absence in nine patients from five families with R133C suggests that its relationship with the Notch3 mutation is coincidental.