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PubMed2005

SNaPshot typing of mitochondrial DNA coding region variants.

We describe a rapid and robust assay to genotype mitochondrial deoxyribonucleic acid (mtDNA) coding region single nucleotide polymorphism (SNPs) using the SNaPshot (Applied Biosystems, Foster City, CA) minisequencing reaction kit. A protocol for mtDNA SNaPshot typing is described in detail, although we emphasize that this method allows great flexibility in the implementation of whatever set of mtDNA SNPs. We discuss the utility of our selection of mtDNA SNPs for molecular anthropologists and forensic geneticists. Firstly, these SNPs allow allocating common mitochondrial West Eurasian haplotypes into their corresponding branches of the mtDNA skeleton, with special attention to the subdivision of sequences belonging to haplogroup H, the most frequent European haplogroup (40-50%) and the worst phylogenetically characterized in the first and second hypervariable segments (HVS-I/II; by far, the most common segments analyzed by sequencing). Second, the polymorphic positions selected for this multiplex reaction considerably increase the discrimination power of current mitochondrial analysis in the forensic field. The method shows high accuracy and robustness, avoiding both the use of alternative time-consuming classical strategies (i.e., restriction fragment length polymorphism typing) and the requirement of high quantities of DNA template.

DNA, Mitochondrial↗
PubMed1998

Novel Mitochondrial DNA Variant That May Give a False Positive Diagnosis for the T8993C Mutation.

Background: Two of the most common mutations in the mitochondrial DNA (mtDNA) of children occur at nucleotide 8993 (nt8993). The base substitutions of T to G (T8993G) and T to C (T8993C) are known to cause neurologic disorders and are routinely screened for in patients suspected of having a mitochondrial disorder. Methods and Results: Both mutations at nt8993 create a novel HpaII restriction endonuclease site and are usually detected by polymerase chain reaction (PCR) amplification of a section of the mtDNA containing nt8993, followed by HpaII digestion. The resulting fragment sizes are then analyzed by agarose gel electropho resis. Initial testing on a child referred for analysis suggested that the proband and his maternal relatives all had the common mtDNA mutation T8993C; however, subsequent restriction endonuclease and DNA sequencing analysis showed that the proband and his maternal relatives were homoplasmic for a novel variant at nt8856. This variant also creates an Hpa II restriction endonuclease site, and the fragments generated by the site are almost identical in size to those generated as a result of the nt8993 mutation when commonly used primers amplify the PCR product. Conclusions: A novel mutation in the mtDNA at nt8856 creates an HpaII restriction endonuclease site that has the potential to generate false positives when PCR products are tested for mutations at nt8993. This emphasizes the need for restriction endonuclease-based diagnostic tests for mtDNA mutations to account for the highly polymorphic nature of the mtDNA sequence and the importance of confirming a mutation by a second method.

Journal Article↗
PubMed2003

Ancestral polymorphisms in genetic markers obscure detection of evolutionarily distinct populations in the endangered Florida grasshopper sparrow (Ammodramus savannarum floridanus).

Genetic analyses of bird subspecies designated as conservation units can address whether they represent units with independent evolutionary histories and provide insights into the evolutionary processes that determine the degree to which they are genetically distinct. Here we use mitochondrial DNA control region sequence and six microsatellite DNA loci to examine phylogeographical structure and genetic differentiation among five North American grasshopper sparrow (Ammodramus savannarum) populations representing three subspecies, including a population of the endangered Florida subspecies (A. s. floridanus). This federally listed taxon is of particular interest because it differs phenotypically from other subspecies in plumage and behaviour and has also undergone a drastic decline in population size over the past century. Despite this designation, we observed no phylogeographical structure among populations in either marker: mtDNA haplotypes and microsatellite genotypes from floridanus samples did not form clades that were phylogenetically distinct from variants found in other subspecies. However, there was low but significant differentiation between Florida and all other populations combined in both mtDNA (FST = 0.069) and in one measure of microsatellite differentiation (theta = 0.016), while the non-Florida populations were not different from each other. Based on analyses of mtDNA variation using a coalescent-based model, the effective sizes of these populations are large (approximately 80,000 females) and they have only recently diverged from each other (< 26,000 ybp). These populations are probably far from genetic equilibrium and therefore the lack of phylogenetic distinctiveness of the floridanus subspecies and minimal genetic differentiation is due most probably to retained ancestral polymorphism. Finally, levels of variation in Florida were similar to other populations supporting the idea that the drastic reduction in population size which has occurred within the last 100 years has not yet had an impact on levels of variation in floridanus. We argue that despite the lack of phylogenetic distinctiveness of floridanus genotypes the observed genetic differentiation and previously documented phenotypic differences justify continued designation of this subspecies as a protected population segment.

Animals↗
PubMed2005

Mitochondrial DNA control region sequences in Koreans: identification of useful variable sites and phylogenetic analysis for mtDNA data quality control.

We have established a high-quality mtDNA control region sequence database for Koreans. To identify polymorphic sites and to determine their frequencies and haplotype frequencies, the complete mtDNA control region was sequenced in 593 Koreans, and major length variants of poly-cytosine tracts in HV2 and HV3 were determined in length heteroplasmic individuals by PCR analysis using fluorescence-labeled primers. Sequence comparison showed that 494 haplotypes defined by 285 variable sites were found when the major poly-cytosine tract genotypes were considered in distinguishing haplotypes, whereas 441 haplotypes were found when the poly-cytosine tracts were ignored. Statistical parameters indicated that analysis of partial mtDNA control region which encompasses the extended regions of HV1 and HV2, CA dinucleotide repeats in HV3 and nucleotide position 16497, 16519, 456, 489 and 499 (HV1ex+HV2ex+HV3CA+5SNPs) and the analysis of another partial mtDNA control region including extended regions of HV1 and HV2, HV3 region and nucleotide position 16497 and 16519 (HV1ex+HV2ex+HV3+2SNPs) can be used as efficient alternatives for the analysis of the entire mtDNA control region in Koreans. Also, we collated the basic informative SNPs, suggested the important mutation motifs for the assignment of East Asian haplogroups, and classified 592 Korean mtDNAs (99.8%) into various East Asian haplogroups or sub-haplogroups. Haplogroup-directed database comparisons confirmed the absence of any major systematic errors in our data, e.g., a mix-up of site designations, base shifts or mistypings.

Asian People↗
PubMed2002

Mitochondrial genome of the colorless green alga Polytomella parva: two linear DNA molecules with homologous inverted repeat Termini.

Most of the well-characterized mitochondrial genomes from diverse green algal lineages are circular mapping DNA molecules; however, Chlamydomonas reinhardtii has a linear 15.8 kb unit mitochondrial genome with 580 or 581 bp inverted repeat ends. In mitochondrial-enriched fractions prepared from Polytomella parva (=P. agilis), a colorless, naturally wall-less relative of C. reinhardtii, we have detected two linear mitochondrial DNA (mtDNA) components with sizes of 13.5 and 3.5 kb. Sequences spanning 97% and 86% of the 13.5- and 3.5-kb mtDNAs, respectively, reveal that these molecules contain long, at least 1.3 kb, homologous inverted repeat sequences at their termini. The 3.5-kb mtDNA has only one coding region (nad6), the functionality of which is supported by both the relative rate at which it has accumulated nonsynonymous nucleotide substitutions and its absence from the 13.5-kb mtDNA which encodes nine genes (i.e., large and small subunit rRNA [LSU and SSU rRNA] genes, one tRNA gene, and six protein-coding genes). On the basis of DNA sequence data, we propose that a variant start codon, GTG, is utilized by the P. parva 13.5-kb mtDNA-encoded gene, nad5. Using the relative rate test with Chlamydomonas moewusii (=C. eugametos) as the outgroup, we conclude that the nonsynonymous nucleotide substitution rate in the mitochondrial protein-coding genes of P. parva is on an average about 3.3 times that of the C. reinhardtii counterparts.

Animals↗
PubMed2002

Progressive external ophthalmoplegia and multiple mitochondrial DNA deletions.

Progressive external ophthalmoplegia (PEO) with secondary accumulation of multiple deletions of mitochondrial DNA (mtDNA) clinically resembles disorders due to primary mutations of mtDNA but follows a Mendelian inheritance pattern. The disorder belongs to an interesting group of diseases in which both the nuclear and the mitochondrial genome are involved in the pathology. Both autosomal dominant (adPEO) and recessive (arPEO) variants of this disorder occur. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients may have multiple mtDNA deletions and/or depletion of mtDNA. Recent reports of mutations in Thymidine Phosphorylase in MNGIE, and of mutations in adenine nucleotide translocator (ANT1), Twinkle and mitochondrial DNA polymerase gamma (POLG) in adPEO, have lead to new insights in the pathogenesis of these disorders of mtDNA maintenance. We also identified POLG mutations in two families with arPEO, which underlines the crucial role of the mtDNA replication machinery for mtDNA maintenance.

Chromosomes, Human, Pair 15↗
PubMed1997

A high observed substitution rate in the human mitochondrial DNA control region.

The rate and pattern of sequence substitutions in the mitochondrial DNA (mtDNA) control region (CR) is of central importance to studies of human evolution and to forensic identity testing. Here, we report a direct measurement of the intergenerational substitution rate in the human CR. We compared DNA sequences of two CR hypervariable segments from close maternal relatives, from 134 independent mtDNA lineages spanning 327 generational events. Ten substitutions were observed, resulting in an empirical rate of 1/33 generations, or 2.5/site/Myr. This is roughly twenty-fold higher than estimates derived from phylogenetic analyses. This disparity cannot be accounted for simply by substitutions at mutational hot spots, suggesting additional factors that produce the discrepancy between very near-term and long-term apparent rates of sequence divergence. The data also indicate that extremely rapid segregation of CR sequence variants between generations is common in humans, with a very small mtDNA bottleneck. These results have implications for forensic applications and studies of human evolution.

Animals↗
PubMed1986

Large mitochondrial genome and mitochondrial DNA size polymorphism in the mosquito parasite, Romanomermis culicivorax.

Physical characterization of the mitochondrial genome derived from the obligate mosquito parasite, Romanomermis culicivorax has generated some surprising physical properties regarding the molecular structure of nematode mitochondrial DNA (mtDNA). Restriction enzyme analysis of this mtDNA has revealed a mitochondrial genome size of approximately 26 kb, the largest metazoan mtDNA reported to date. Isofemale lineages are monomorphic for one of three size variants, differing by 500-1,000 base pairs, present in our original field population. Cloned hybridization probes derived from a single region exhibiting a 600 bp size polymorphism share strong homology with several spatially separated sites distributed about the mtDNA. This suggests that the homology is a result of repeated DNA sequence elements contained within this mitochondrial genome that contribute to mtDNA size polymorphism.

Animals↗
PubMed2004

Interaction among different heterocromatic variants in the grasshopper Dichroplus elongatus.

Simultaneous chromosome polymorphisms for supernumerary elements allow us to analyse the relationships among different forms of heterochromatic variation in nature. We report simultaneous variation patterns for supernumerary segments in chromosomes S10 (SS10), S9 (SS9) and S6 (SS6) and B chromosomes in nine populations of the grasshopper Dichroplus elongatus from two biogeographic provinces from east Argentina. Our results show spatial chromosome differentiation for three out of four supernumerary heterochromatic variants (B chromosomes, SS6 and SS10). The incidence of B chromosomes was negatively correlated with the SS10 frequency. The distribution pattern analysis shows different degree of differentiation among populations for each supernumerary heterochromatic variant suggesting that the detected chromosome variation cannot be explained by interaction between migration and genetic drift. Moreover, the observed population chromosome differentiation was not in agreement with the hierarchical analysis of molecular of heterogeneity at mitochondrial DNA level (mtDNA). The present results point out the importance of the interaction among heterochromatic variants in the chromosome intraspecific variation in east Argentina natural populations of the grasshopper D. elongatus.

Animals↗
PubMed2005

Comparative analysis of the pathogenic mechanisms associated with the G8363A and A8296G mutations in the mitochondrial tRNA(Lys) gene.

Two mutations (G8363A and A8296G) in the mtDNA (mitochondrial DNA) tRNA(Lys) gene have been associated with severe mitochondrial diseases in a number of reports. Their functional significance, however, remains unknown. We have already shown that homoplasmic cybrids harbouring the A8296G mutation display normal oxidative phosphorylation, although the possibility of a subtle change in mitochondrial respiratory capacity remains an open issue. We have now investigated the pathogenic mechanism of another mutation in the tRNA(Lys) gene (G8363A) by repopulating an mtDNA-less human osteosarcoma cell line with mitochondria harbouring either this genetic variant alone or an unusual combination of the two mutations (A8296G+G8363A). Cybrids homoplasmic for the single G8363A or the A8296G+G8363A mutations have defective respiratory-chain enzyme activities and low oxygen consumption, indicating a severe impairment of the oxidative phosphorylation system. Generation of G8363A cybrids within a wild-type or the A8296G mtDNA genetic backgrounds resulted in an important alteration in the conformation of the tRNA(Lys), not affecting tRNA steady-state levels. Moreover, mutant cybrids have an important decrease in the proportion of amino-acylated tRNA(Lys) and, consequently, mitochondrial protein synthesis is greatly decreased. Our results demonstrate that the pathogenicity of the G8363A mutation is due to a change in the conformation of the tRNA that severely impairs aminoacylation in the absence of changes in tRNA stability. The only effect detected in the A8296G mutation is a moderate decrease in the aminoacylation capacity, which does not affect mitochondrial protein biosynthesis.

Aminoacylation↗
PubMed1993

Phylogeny and historical aspects of the ecology of eastern Australian scrubwrens Sericornis spp.--evidence from mitochondrial DNA.

A phylogeny of mainland eastern Australian scrubwrens (Sericornis citreogularis, S. frontalis, S. keri, S. magnirostris and S. beccarii) was constructed using presence or absence of phylogenetically informative restriction sites in 15 mitochondrial DNA (mtDNA) haplotypes. MtDNA sequence divergences between species were generally large (mostly between 10 and 18%) emphasizing the antiquity of this radiation. Phylogenetic analysis of the mtDNA resulted in a strongly supported phylogeny concordant with relationships suggested by a previous study of allozyme variation, but conflicting with concepts based on morphological similarity. The mtDNAs grouped the species into two lineages, one containing beccarii and magnirostris and the other comprising citreogularis, frontalis and keri. The mtDNAs of beccarii and magnirostris were paraphyletic with a northern variant of magnirostris being more similar to beccarii than other magnirostris. The mtDNA from keri was most closely related to that of frontalis and then citreogularis. The strongly corroborated molecular phylogeny confirms and extends the previous (Christidis et al. 1988; Auk 105: 616) reinterpretation of morphological and ecological shifts in the group. This suggests considerable plasticity and parallelism in the plumage characters previously used to associate species. The low divergence and close relationship between the montane rain-forest specialist keri and the widely distributed generalist frontalis demonstrate the capacity for evolutionarily rapid and dramatic shifts in habitat use. This contrasts with the assumption of fixed habitat preference that underlies refuge-based models of diversification of rain-forest fauna.

Animals↗
PubMed2003

Molecular characterization of WFS1 in patients with Wolfram syndrome.

Wolfram (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness) syndrome is a rare autosomal-recessive neurodegenerative disorder that is characterized by juvenile-onset diabetes mellitus, optic atrophy, diabetes insipidus, and sensorineural hearing impairment. A gene responsible for Wolfram syndrome (WFS1) has been identified on the short arm of chromosome 4 and subsequently mutations in WFS1 have been described. We have screened 12 patients with Wolfram syndrome from nine Dutch families for mutations in the WFS1-coding region by single-strand conformation polymorphism analysis and direct sequencing. Furthermore, we analyzed the mitochondrial genome for gross abnormalities and the A3243G point mutation in the leucyl-tRNA gene, because Wolfram syndrome shows phenotypic similarities with mitochondrial disease. Seven mutations in WFS1 were identified in six of nine families: two missense mutations, one frameshift mutation, one splice donor site mutation, and three deletions. In addition, a splice variant near the 5'UTR of WFS1 was identified, present in patient as well as control RNA samples in various percentages, alternating the translation initiation consensus sequence. Whether this WFS1 splice variant displays impaired translation efficiency remains to be determined. No MtDNA lesions were identified in any of the Wolfram patients. Our results demonstrate the usefulness of molecular analysis of WFS1 in the refinement of clinical diagnostic criteria for Wolfram syndrome that helps to dissect the clinically overlapping syndromes sharing diabetes mellitus and optic atrophy.

Adolescent↗
PubMed1974

Mitochondrial DNA replication in sea urchin oocytes.

Mitochondrial DNA (mtDNA) replicative intermediates from Strongylocentrotus purpuratus oocytes were isolated by ethidium bromide-CsCl density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the mtDNA was radioactively labeled by exposing isolated oocytes to [(3)H]thymidine. Oocyte mtDNA replication appears to follow the displacement loop model outlined in mouse L cells. There are differences in detail. The frequency of D-loop DNA is much lower in oocytes, suggesting that the relative holding time at the D-loop stage is shorter. Duplex synthesis on the displaced strand occurs early and with multiple initiations. The frequency of totally duplex replicative forms, or Cairns' forms, is the highest reported for mtDNA. The differences may be related to the fact that oocyte mtDNA replication occurs in the absence of cell division and need not be coordinated with a cell cycle. Molecules with expanded D loops banded in the intermediate region between the lower and upper bands in an ethidium bromide-CsCl gradient, supporting the notion that displacement replication proceeds on a closed circular template which is subject to nicking-closing cycles. In mature sea urchin eggs, replicative forms are absent and virtually all the mtDNA is stored as clean circular duplexes. Some novel structural variants of superhelical circular DNA (molecules with denaturation loops and double branch-migrated replicative forms) are reported.

Animals↗
PubMed2006

The novel A4435G mutation in the mitochondrial tRNAMet may modulate the phenotypic expression of the LHON-associated ND4 G11778A mutation.

PURPOSE: To investigating the role of mitochondrial haplotypes in the development of Leber's hereditary optic neuropathy (LHON) associated with the ND4 G11778A mutation in Chinese families. METHODS: A three-generation Chinese family with LHON was studied by clinical and genetic evaluation as well as molecular and biochemical analysis of mitochondrial (mt)DNA. RESULTS: This family exhibits a high penetrance and expressivity of visual impairment. The average age at onset was 13.9 years in this family. Of the family members, 86% of the male and 29% of the female matrilineal relatives had visual loss, with a wide range of severity, from blindness to nearly normal vision. Molecular analysis of mtDNA identified the homoplasmic ND4 G11778A mutation and 35 other variants, belonging to the Asian haplogroup D5. Of other variants, the novel homoplasmic A4435G mutation absent in 164 Chinese controls is localized at 3' end adjacent to the anticodon, at conventional position 37 (A37), of tRNAMet. The adenine (A37) at this position of tRNAMet is extraordinarily conserved from bacteria to human mitochondria. This modified A37 was shown to contribute to the high fidelity of codon recognition and to the structural formation and stabilization of functional tRNAs. In fact, the significant reduction of the steady state levels in tRNAMet was observed in cells carrying the both the A4435G and G11778A mutations but not cells carrying only the G11778A mutation. Thus, a failure in mitochondrial tRNA metabolism, caused by the A4435G mutation, may worsen the mitochondrial dysfunction associated with the primary G11778A mutation. CONCLUSIONS: The novel tRNAMet A4435G mutation has a potential modifier role in increasing the penetrance and expressivity of the primary LHON-associated G11778A mutation in this Chinese family.

Adolescent↗
PubMed1998

[Variability of mitochondrial DNA in three groups of indigenous inhabitants of Northern Asia].

Sequence variants of the hypervariable region I of human mitochondrial DNA (mtDNA) of 125 individuals from three aboriginal population groups of northern Asia (Yakuts, Evens, and Koryaks) were analyzed. Unique types of mtDNA (mitotypes) were discovered in 80% of Koryaks, 78% of Evens, and 59% of Yakuts. The mitotypes observed were clustered into nine phylogenetically related groups, two of which, according to the data on the comparative analysis of Siberian and east Asian populations, were Koryak-specific, and one was Even-specific. Koryaks and Evens exhibited mtDNAs that were highly frequent in Ainu. The results are discussed in terms of genetic differentiation and the ethnogenesis of ethnic groups of northern Asia.

Asia↗
PubMed1995

The origins of the Polynesians: an interpretation from mitochondrial lineage analysis.

Using mitochondrial lineage analysis of 1,178 individuals from Polynesia, the western Pacific, and Taiwan, we show that the major prehistoric settlement of Polynesia was from the west and involved two or possibly three genetically distinct populations. The predominant lineage group, accounting for 94% of Polynesian mtDNA, shares a 9-bp COII/tRNA(Lys) intergenic deletion and characteristic control region transition variants, compared to the Cambridge reference sequence. In Polynesia, the diversity of this group is extremely restricted, while related lineages in Indonesia, the Philippines, and Taiwan are increasingly diverse. This suggests a relatively recent major eastward expansion into Polynesia, perhaps originating from Taiwan, in agreement with archeological and linguistic evidence, but which experienced one or more severe population bottlenecks. The second mitochondrial lineage group, accounting for 3.5% of Polynesian mtDNA haplotypes, does not have the 9-bp deletion and its characterized by an A-C transversional variant at nt position 16265. Specific oligonucleotides for this variant were used to select individuals from the population sample who, with other sequences, show that the Polynesian lineages were part of a diverse group in Vanuatu and Papua New Guinea. The very low overall diversity of both lineage groups in Polynesia suggests there was severe population restriction during the colonization of remote Oceania. A third group, represented by only four individuals (0.6%) in Polynesia but also present in the Philippines, shares variants at nt positions 16172 and 16304. Two Polynesians had unrelated haplotypes matching published sequences from native South Americans, which may be the first genetic evidence of prehistoric human contact between Polynesia and South America.

Base Sequence↗
PubMed2002

[Implication of single nucleotide polymorphisms in association study: mitochondrial variations as another genetic markers for hypertension].

Although multiple nuclear gene polymorphisms have been identified as potential risk factors for hypertension, the linkage between extranuclear DNA variations and hypertension has been uncertain. We investigated whether mitochondrial DNA(mtDNA) polymorphisms are implicated in Japanese hypertension. We used direct sequencing methods to search for single nucleotide polymorphisms(SNPs) in a hypervariable segment of the mitochondrial control region in each blood sample from 20 hypertensives and 20 normotensives. Then, we determined the distribution of two SNPs, T16223C and C16362T, in 183 hypertensives and 193 healthy subjects in the Aomori population in the northern area of Honshu island of Japan. The relationship between the gene polymorphism and hypertension was evaluated using chi-square test. Seventy SNPs were found there and the number of SNPs in each individual was significantly greater(p = 0.0111) in hypertensives than in normotensives. The C16223 genotype was more frequent in hypertensives than in normotensives(p = 0.0018). There was no significant difference in C16362T variant frequency between the groups. From these results, we conclude that mtDNA SNPs were enriched in Japanese hypertension and that the mtDNA C16223 genotype may be one of the genetic susceptibility factors for hypertension.

DNA, Mitochondrial↗
PubMed2006

A reappraisal of complete mtDNA variation in East Asian families with hearing impairment.

In a number of recent studies, we summarized the obvious errors and shortcomings that can be spotted in many (if not most) mitochondrial DNA (mtDNA) data sets published in medical genetics. We have reanalyzed here the complete mtDNA genome data published in various recent reports of East Asian families with hearing impairment, using a phylogenetic approach, in order to demonstrate the persistence of lab-specific mistakes in mtDNA genome sequencing in cases where those caveats were (deliberately) neglected. A phylogenetic reappraisal of complete mtDNAs with mutation A1555G (or G11778A) indeed supports the suggested lack of association between haplogroup background and phenotypic presentation of these mutations in East Asians. In contrast, the claimed pathogenicity of mutation T1095C in Chinese families with hearing impairment seems unsupported, basically because this mutation is rather basal in the mtDNA phylogeny, being specific to haplogroup M11 in East Asia. The roles of other haplogroup specific or associated variants, such as A827G, T961C, T1005C, in East Asian subjects with aminoglycoside-induced and non-syndromic hearing loss are also unclear in view of the known mtDNA phylogeny.

DNA, Mitochondrial↗