A 9-bp Deletion Homoplasmy in Women with Polycystic Ovary Syndrome Revealed by Mitochondrial Genome-Mutation Screen

Polycystic ovary syndrome (PCOS) is a complex and heterogeneous disorder presenting a challenge for clinical investigators. To investigate the association of a mitochondrial genetic basis with PCOS, we screened mutations of the whole mitochondrial genome in 57 women patients with PCOS and 38 healthy control individuals. Two-step PCR reactions were adopted to amplify and sequence the whole mitochondrial genome. A 9-bp deletion variant appeared in homoplasmy between PCOS patients and control individuals. In the 62 individuals with complete sequences, eight of 34 (23.5%) patients showed the 9-bp deletion, compared with only two of 28 (7.1%) in healthy controls. The 9-bp deletion variant in region V of mitochondrial DNA may be associated with the heterogeneous disorder PCOS.     

A Russian family of Slavic origin carrying mitochondrial DNA with a 9-bp deletion in region V and a long C-stretch in D-loop

A 9-bp deletion first described in the mitochondrial DNA (mtDNA) for East Asian, Polynesian or Indian American populations of the B haplogroup is now discovered in Slavs. The Russian family carrying that deletion belongs to a new branch of the T haplogroup as deduced from D-loop sequence and haplogroup-specific restriction fragment length polymorphism analysis. One family member had a Kearns-Sayre syndrome with a 5.5 kb mtDNA deletion. This family also presented a long C-stretch in the D-loop. Whether or not the formation of the 5.5 kb deletion might be related to the 9-bp deletion or to the long C-stretch in the D-loop is discussed.     

Insertion–Deletion Polymorphism of Mitochondrial DNA Region V in Kazakh Populations from Different Regions of Kazakhstan

Insertion–deletion polymorphism of the mitochondrial DNA (mtDNA) region V was examined in three Kazakh populations inhabiting different regions of Kazakhstan. The 9-bp deletion was revealed in all three populations examined. In Altai population the 4-bp insertion was also found. The presence of these polymorphic variants was confirmed by DNA sequencing.     

Absence of the 9-bp deletion of mitochondrial DNA in pre-Hispanic inhabitants of Argentina

We investigated the incidence of the Region V mitochondrial DNA 9-base-pair (bp) deletion from human remains recovered from several archaeological sites and contexts throughout Argentina. Of the 34 samples analyzed, 24 yielded DNA extractions that gave clear amplification results. All of the individuals carried two repeats of the 9 bp, one of which has been shown to be deleted in some individuals of Asian origin and defines mitochondrial lineage B. Although most of the modern Amerindian groups in the region exhibit the deletion in high frequencies, the absence of the 9-bp deletion among ancient populations of South America seems to be the rule rather than the exception, as was reported by several studies involving extinct populations. The evidence gathered until now suggests that the earliest settlers of this region of South America did not carry mitochondrial lineage B.     

Evolutionary history of the COII/tRNALys intergenic 9 base pair deletion in human mitochondrial DNAs from the Pacific

Length changes in human mitochondrial DNA (mtDNA) are potentially useful markers for inferring the evolutionary history of populations. One such length change is a nine base pair (9-bp) deletion that is located in the intergenic region between the COII gene and the Lysine tRNA gene (COII/tRNALys intergenic region). This deletion has been used as a genetic marker to trace descent from peoples of East Asian origin. A geographic cline of the deletion frequency across modern Pacific Islander populations suggests that the deletion may be useful for tracing prehistoric Polynesian origins and affinities. Mitochondrial DNA sequence variation within two variable segments of the control region (CR) permits a number of inferences regarding the evolutionary history of the 9-bp deletion that cannot be determined from frequency data alone. We obtained CR sequences from 74 mtDNAs with the 9-bp deletion from Indonesia, coastal Papua New Guinea (PNG), and American Samoa. Phylogenetic and pairwise distribution analysis of these CR sequences pooled with previously published CR sequences reveals that the deletion arose independently in Africa and Asia and suggests possible multiple origins of the deletion in Asia. A clinal increase of the frequency of the 9-bp deletion across the three Pacific populations is associated with a decrease in CR sequence diversity, consistent with founder events. Furthermore, analysis of pairwise difference distributions indicates an expansion time of proto-Polynesians that began 5,500 yr ago from Southeast Asia. These results are consistent with the express train model of Polynesian origins.      

Insertion-deletion polymorphism of mitochondrial DNA region V in Kazakh populations from different regions of Kazakhstan

Insertion-deletion polymorphism of the mitochondrial DNA (mtDNA) region V was examined in three Kazakh populations inhabiting different regions of Kazakhstan. The 9-bp deletion was revealed in all three populations examined. In Altai population the 4-bp insertion was also found. The presence of these polymorphic variants was confirmed by DNA sequencing.     

Mitochondrial DNA variation is an indicator of austronesian influence in Island Melanesia.

Past studies have shown a consistent association of a specific set of mitochondrial DNA 9 base pair (bp) deletion haplotypes with Polynesians and their Austronesian-speaking relatives, and the total lack of the deletion in a short series of New Guinea Highlanders. Utilizing plasma and DNA samples from various old laboratory collections, we have extended population screening for the 9-bp deletion into "Island Melanesia," an area notorious for its extreme population variation. While the 9-bp deletion is present in all Austronesian, and many non-Austronesian-speaking groups, it is absent in the more remote non-Austronesian populations in Bougainville and New Britain. These results are consistent with the hypothesis that this deletion was first introduced to this region about 3,500 years ago with the arrival of Austronesian-speaking peoples from the west, but has not yet diffused through all populations there. The pattern cannot be reconciled with the competing hypothesis of a primarily indigenous Melanesian origin for the ancestors of the Polynesians. Although selection clearly has operated on some other genetic systems in this region, both migration and random genetic drift primarily account for the remarkable degree of biological diversity in these small Southwest Pacific populations.     

The mutation rate of the human mtDNA deletion mtDNA4977.

The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.     

The age of a unique event polymorphism

We develop a Markov chain Monte Carlo approach for estimating the distribution of the age of a mutation that is assumed to have arisen just once in the history of the population of interest. We assume that in addition to the presence or absence of this mutation in a sample of chromosomes, we have DNA sequence data from a region completely linked to the mutant site. We apply our method to a mitochondrial data set in which the DNA sequence data come from hypervariable region I and the mutation of interest is the 9-bp region V deletion.     

A topoisomerase II cleavage site is associated with a novel mitochondrial DNA deletion

Mitochondrial myopathies and encephalopathies can be caused by nucleotide substitutions, deletions or duplications of the mitochondrial DNA (mtDNA). In one such disorder, Kearns-Sayre Syndrome (KSS), large-scale hetero-plasmic mtDNA deletions are often found. We describe a 14-year-old boy with clinical features of KSS, plus some additional features. Analysis of the entire mitochondrial genome by the polymerase chain reaction and Southern blotting revealed a 7864-bp mtDNA deletion, heteroplasmic in its tissue distribution. DNA sequencing established that the deletion was between nucleotides 6238 and 14103, and flanked by a 4-bp (TCCT) direct repeat sequence. Deletions between direct repeats have been hypothesised to occur by a slipped-mismatching or illegitimate recombination event, or following the DNA cleavage action of topoisomerase II. Analysis of the gene sequence in the region surrounding the mtDNA deletion breakpoint in this patient revealed the presence of putative vertebrate topoisomerase II sites. We suggest that direct repeat sequences, together with putative topoisomerase II sites, may predispose certain regions of the mitochondrial genome to deletions.     

Direct sequencing of deleted mitochondrial DNA in myopathic patients

To investigate the mechanism of mitochondrial DNA deletion in human diseases, we amplified the deleted mitochondrial DNA of five patients with mitochondrial myopathy by using the polymerase chain reaction, and directly sequenced the crossover regions of the deleted mitochondrial DNA without cloning. In Patient 1, a 7-bp directly repeated sequence of 5'-ATCCCCA-3' was found at the boundaries of deleted segment spanning 7,039 bp between the ATPase 6 and the cytochrome b genes. In Patients 2, 3, and 4, a 13-bp sequence of 5'-ACCTCCCTCACCA-3' was found in the boundaries of deleted segment spanning 4,977 bp between the ATPase 8 and the ND5 genes. In Patient 5, a 3-bp sequence of 5'-CCT-3' was found in the boundaries of deleted segment spanning 3,717 bp between the ATPase 6 and the ND5 genes. Similar directly repeated sequences may contribute to mitochondrial DNA deletions in human degenerative diseases.     

Palindromy and the Location of Deletion Endpoints in Escherichia Coli

The contributions of direct and inverted repeats to deletion formation were studied by characterizing Ampr revertants of plasmids with a series of insertion mutations at a specific site in the pBR322 ampicillin resistance (amp) gene. The inserts at this site are palindromic, variable in length, and bracketed by 9- or 10-bp direct repeats of amp sequence. There is an additional direct repeat composed of 4 bp within the insert and 4 bp of adjoining amp sequence. DNA sequencing and colony hybridization of Ampr revertants showed that they contained either the parental amp sequence, implying deletion endpoints in the flanking 9- or 10-bp repeats, or a specific 1-bp substitution, implying endpoints in the 4-bp repeats. Although generally direct repeats seem to be used as deletion endpoints with a frequency proportional to their lengths, we found that with uninterrupted palindromes longer than 32 bp, the majority of deletions ended in the 4 bp, not the 9- or 10-bp repeats. This preferential use of the shorter direct repeats associated with palindromes is interpreted according to a DNA synthesis-error model in which hairpin structures formed by intrastrand pairing foster the slippage of nascent strands during DNA synthesis.     

Absence of the Asian-specific region V mitochondrial marker in Native Beringians.

The Asian-specific 9-bp deletion between the genes for mitochondrial cytochrome oxidase II and lysine transfer RNA has been used to trace aboriginal human movements out of Southeast Asia and into portions of the South Pacific. Although it has been used to estimate the number of independent lineages that occur in the New World, it has not been studied in native peoples of the Beringian region. Thus, we have used PCR to amplify and compare the lengths of DNA segments surrounding this deletion in native peoples of Beringia and the adjacent regions, as well as natives of the Altai Mountains of Southwestern Siberia. Of the 176 individuals analyzed here, the deletion was found in only 3 of 25 individuals from the Ust-Kan region of the Altai Mountains. We comment on the distribution of this marker and on potential relationships between Beringians and other Native American groups in which this marker has been surveyed. One Chukchi possessed three copies of the 9-bp sequence, which suggests (1) that the number of copies of this sequence in humans may be more variable than had been believed and (2) that a mechanism of replication based on tandem duplication may be a potential explanation for the origin of this length mutation in humans.     

Ageing-associated 5 kb deletion in human liver mitochondrial DNA

Using PCR technique and restriction mapping, we analyzed liver mitochondrial DNA (mtDNA) of 2 stillborn babies and 55 Chinese subjects from 27 to 86 years old and blood cell mtDNA from 20 subjects of various ages. An ageing-associated 4,977-bp deletion was detected between nucleotide position 8,469 and 13,447 (or between 8,482 and 13,460) in the liver mtDNA of older subjects. In the region containing the junction fragment, we observed a 13 bp repeat "ACCTCCCTCACCA". Moreover, the incidence of the deleted mtDNA of each of the study subjects was found to increase with age. The deletion was found in 5 out of 8 patients of the 31-40 age group and 9 out of 11 patients of the 41-50 age group, and in all the patients over 50 years old. The deletion was not observed in either the mtDNA of the liver of the stillbirth or the blood cells of subjects of all the age groups. These results support our previous contention that liver mitochondrial respiratory functions decline with age and the hypothesis that continuous accumulation of mitochondrial DNA mutation is an important contributor to ageing process.     

Mitochondrial DNA deletion Δ4977 in peptic ulcer disease

Reactive oxygen species (ROS) play a critical role in peptic ulcer disease (PUD). Due to the high rate of ROS production and limited capacity for DNA repair within mitochondria, mtDNA is susceptible to oxidative damage and mutations. mtDNA deletion Δ4977 is one of the most common deletion events identified in mitochondria. We examined the association of 4977-bp mtDNA deletion with PUD. Genotypes were determined in bioptic samples of 150 PUD patients and 190 controls. The 4977-bp mtDNA deletion was found more frequently among patients with PUD (52%) than among controls (22.63%). The strong association between the mtDNA 4977-bp deletion and PUD was confirmed (OR = 3.7; 95% CI, 2.32–5.91; P = 0.0001). The 4977-bp deletion in mitochondrial DNA may be a risk factor for PUD, or may reflect an increase in oxidative stress that commonly accompanies underlying PUD disease. Larger population-based studies are needed to uncover the possible causal relationship between this deletion and peptic ulcer disease.     

Mitochondrial DNA variability of West New Guinea populations.

This paper reports human mitochondrial DNA variability in West New Guinea (the least known, western side of the island of New Guinea), not yet described from a molecular perspective. The study was carried out on 202 subjects from 12 ethnic groups, belonging to six different Papuan language families, representative of both mountain and coastal plain areas. Mitochondrial DNA hypervariable region 1 (HVS 1) and the presence of the 9-bp deletion (intergenic region COII-tRNA(Lys)) were investigated. HVS 1 sequencing identified 73 polymorphic sites defining 89 haplotypes; the 9-bp deletion, which is considered a marker of Austronesian migration in the Pacific, was found to be absent in the whole West New Guinea study sample. Statistical analysis applied to the resulting haplotypes reveal high heterogeneity and an intersecting distribution of genetic variability in these populations, despite their cultural and geographic diversity. The results of subsequent phylogenetic approaches subdivide mtDNA diversity in West New Guinea into three main clusters (groups I-III), defined by sets of polymorphisms which are also shared by some individuals from Papua New Guinea. Comparisons with worldwide HVS 1 sequences stored in the MitBASE database show the absence of these patterns outside Oceania and a few Indonesian subjects, who also lack the 9-bp deletion. This finding, which is consistent with the effects of genetic drift and prolonged isolation of West New Guinea populations, lead us to regard these patterns as New Guinea population markers, which may harbor the genetic memory of the earliest human migrations to the island.     

Detection and characterization of mitochondrial DNA rearrangements in Pearson and Kearns-Sayre syndromes by long PCR

We used a strategy based on long PCR (polymerase chain reaction) for detection and characterization of mitochondrial DNA (mtDNA) rearrangements in two patients with clinical signs suggesting Pearson syndrome and Kearns-Sayre syndrome (KSS), respectively, and one patient with myopathic symptoms of unidentified origin. Mitochondrial DNA rearrangements were detected by amplification of the complete mitochondrial genome (16.6 kb) using long PCR with primers located in essential regions of the mitochondrial genome and quantified by three-primer PCR. Long PCR with deletion-specific primers was used for identification and quantitative estimation of the different forms of rearranged molecules, such as deletions and duplications. We detected significant amounts of a common 7.4-kb deletion flanked by a 12-bp direct repeat in all tissues tested from the patient with Pearson syndrome. In skeletal muscle from the patient with clinical signs of KSS we found significant amounts of a novel 3.7-kb rearrangement flanked by a 4-bp inverted repeat that was present in the form of deletions as well as duplications. In the patient suffering from myopathic symptoms of unidentified origin we did not detect rearranged mtDNA in blood but found low levels of two rearranged mtDNA populations in skeletal muscle, a previously described 7-kb deletion flanked by a 7-bp direct repeat and a novel 6.6-kb deletion with no repeat. These two populations, however, were unlikely to be the cause of the myopathic symptoms as they were present at low levels (10–40 ppm). Using a strategy based on screening with long PCR we were able to detect and characterize high as well as low levels of mtDNA rearrangements in three patients. Received: 10 March 1997 / Accepted: 20 May 1997     

The 9-bp deletion between the mitochondrial lysine tRNA and COII genes in tribal populations of India

A 9-base-pair (bp) deletion located between the lysine tRNA (MTTK) and COII (MTCOX*2) genes in the human mitochondrial genome is a valuable marker for tracing population relationships. Previous research has shown that the 9-bp deletion is associated with two major clusters of control region sequences; one occurs in sub-Saharan Africa, while the other is associated with Asian populations and populations of Asian origin. We surveyed 898 individuals from 16 tribal populations in India and found 6 individuals with the 9-bp deletion. Sequences of the first hypervariable segment (HV1) of the mtDNA control region from these 9-bp deletion-bearing mtDNAs were compared to those previously reported from Asian and African populations. Phylogenetic analysis indicates three distinct clusters of tribal Indian 9-bp deletion mtDNA types. One cluster, found in northeast India, includes southeast Asian and Indonesian mtDNA types. The remaining two clusters appear to have unique origins in southern India. These data provide further evidence of past migrations from Asia into the northeast corner of the Indian subcontinent.     

Genetic diversity of moose (Alces alces L.) in Eurasia

Polymorphism of nucleotide sequence of D-loop fragment of the mitochondrial DNA was studied in 20 moose from several local populations on the territory of Eurasia. Three main haplotype variants of D-loop were detected by molecular phylogenetic method, which formed three clusters named European, Asian and American. Intraspecies variation in the length of HVSI of D-loop of the mitochondrial DNA of moose was revealed. In the Far Eastern and Yakutian moose, haplotypes with a 75-bp deletion were found, which were most similar with haplotypes (also with the deletion), earlier observed in North American moose [1]. The highest diversity of the haplotypes of mitochondrial DNA is characteristic of Yakutia and the Far East (where three haplotype variants were found), which demonstrates the probable role of the region as the center of the species or as the region of ancient population mixture. The geographic region might be considered as a probable source of ancient moose migrations from Asia to America, basing on the data of distribution of mitochondrial haplotypes of D-loop and alleles of MhcAlal-DRB1. Divergence of nucleotide sequences of haplotypes with the 75-bp deletion (forming the American cluster on the phylogenetic tree) was the lowest (0.4%), which evidences respectively recent origin of the group of haplotypes. In Europe, only haplotypes of mitochondrial DNA referred to European variant were observed. Basing on analysis of variation of nucleotide sequences of D-loop, exon 4 of kappa-Casein and exon 2 of MhcALal-DRB1, we demonstrated that Eurasian moose studied belong to the unique species, which has probably passed through a bottle neck. The time of the origin of modern diversity of D-loop haplotypes of the species was estimated as 0.075-0.15 Myr ago.     

Length variations in the COII-tRNA(Lys) intergenic region of mitochondrial DNA in Indonesian populations.

The prevalence of a 9-base-pair (bp) deletion between the mitochondrial cytochrome oxidase II (MTCOX*2) and lysine tRNA (MTTK) genes (region V) has been used to estimate the genetic relationships among Asian and Pacific populations. Many East Asian and Pacific Island populations have been examined previously, but the mitochondrial DNA (mtDNA) diversity of the intervening Indonesian archipelago has not previously been systematically examined. The 17,500 islands of Indonesia currently contain nearly 213 million people and extensive cultural, linguistic, and, presumably, genetic diversity. This study of 1091 individuals representing 15 ethnic groups is the most extensive mtDNA survey to date of the Indonesian archipelago. Six distinct length polymorphisms in region V were observed within these 15 populations. The 9-bp deletion was found in every population examined at frequencies comparable to those of previously examined East Asian populations and substantially lower than those in most Pacific Island populations. Despite the inclusion of Austronesian-speaking populations and a Papuan-speaking population, there was no statistically significant heterogeneity in the frequency of the 9-bp deletion among the 15 populations (p = 0.09). These data indicate that substantial gene flow occurred among the populations at some time in the past. Our observations of no significant correlations between genetic and geographic distances (r = -0.04, p = 0.53) coupled with the extensive cultural and linguistic differences currently within the archipelago suggest that little gene flow among neighboring populations has occurred recently.