Sequence variations of mitochondrial DNA D-loop region are highly frequent events in familial breast cancer

Mitochondrial DNA (mtDNA) is known for its high frequencies of polymorphisms and mutations. The non-coding displacement (D)-loop, especially a mononucleotide repeat (poly-C) between 303 and 315 nucleotides (D310), has been recently identified as a frequent hotspot of mutations in human neoplasia, including breast cancer. To further explore the sequence variations of mitochondrial D-loop region in familial breast cancer and their possible associations with breast cancer risk, PCR-SSCP and direct DNA sequencing methods were used to detect the variants of the mtDNA D-Loop in 23 familial breast cancer patients as well as three high-risk cancer families. Compared to that in sporadic breast tumors (53.3%, 16/30) and healthy blood donors (6.7%, 2/30), we identified a total of 126 sequence alterations in 23/23 (100%) of familial breast cancer patients, including eight novel nucleotide variants. Among these changes, A to G at nt.263, T to C at nt.489, T to C at nt.310, TC insertion at nt.311, CA deletion at nt.522, and C to G at nt.527 were highly frequent ones. In addition, among three high-risk cancer families, we found that individuals affected with breast cancer harbored more mtDNA sequence variants in mtDNA D310 area than other affected family members. Together, our data indicate that sequence variants within the mtDNA D-Loop region are frequent events in Chinese familial breast cancer patients. Some of these nucleotide abnormalities, particularly those in D310 segment, might be involved in the breast carcinogenesis and could be included in a panel of molecular biomarkers for cancer susceptibility early-detection strategy.     

线粒体基因组D-Loop区基因多态性与2型糖尿病的相关性

随机选取199例浙江地区2型糖尿病患者与102例正常对照, 采用聚合酶链反应(Polymerase chain re-action, PCR)、基因片段直接测序来检测线粒体基因组D-Loop区域基因变异情况, 同时分析其与主要临床指标的关系。结果显示: 线粒体基因组D-Loop区域为一高变异区, np73A-G、np263A-G、np16223C-T、np16519T-C为4个高变异位点; 发现29个未见报道的新变异位点; np193A-G、np234A-G、np16108C-T等变异与糖尿病家族史有关。这表明浙江籍汉族人线粒体基因组D-Loop区存在大量基因多态性现象, 此区域的某些变异可能与糖尿病的发生发展等具有一定相关性。     

重建邻接关系树评估原发性高血压患者的遗传性

探讨原发性高血压患者的遗传性,评估线粒体D环控制区基因变异在高血压发病中的作用。提取原发性高血压患者和正常血压人群DNA各20例,用3对交叉重叠引物扩增全部线粒体控制区D环基因,直接基因测序并重建邻接关系树,分析原发性高血压的基因变异特点。结果发现部分高血压病患者具有明显群聚倾向,与正常血压人群和其他无群聚倾向的高血压患者比较,存在高频率、高密度的D环控制区基因变化（P<0.01）,尤以np152T->C、np182C->T、np189A->G、np247G->A、np16187C->T、np16189T->C、np16264C->T、np16270C->T和np16311T->C等多态性变化显著,并因此造成np16184~16193微卫星区域多聚C长度改变。本研究提示部分高血压病患者有群聚现象,基因型np152C、np182T、np247A、np16187T、np16189C、np16264T、np16270T和np16311C可能是此聚类族高血压患者的易感遗传标记。Abstract：To explore the inheritable character in essential hypertension and to evaluate the role of mitochondrial DNA (mtDNA) variations of the D-loop region in the pathogenesis of hypertension, the entire genome of the D-loop region from the hypertensive and the normotensive (20 cases, each) was amplified using 3 pairs of overlapping primers and followed by sequencing. We reconstructed the Neighbor-Joining tree and analyzed the mtDNA variations in the D-loop region. The results exhibited that one clustering branch harbored some hypertensive, who had significantly higher frequency and density of mtDNA variations (both P<0.01), especially the polymorphisms of np152T->C, np182C->T, np189A->G, np247G->A, np16187C->T, np16189T->C, np16264C->T, np16270C->T and np16311T->C. This study suggested that there was an aggregative phenomenon in some hypertensive. The genotypes of np152C, np182T, np247A, np16187T, np16189C, np16264T, np16270T and np16311C may be potential genetic markers for susceptibility to hypertension.     

人癌细胞线粒体DNA控制区序列特征分析

为了探讨癌细胞mtDNA控制区序列的变化特征, 采用PCR产物限制性片段长度多态性(PCR-RFLP)分析与直接测序相结合的方法,对比分析6株人癌细胞系、 6例癌患者及4例健康成人白细胞mtDNA控制区序列。发现第16519位T→C、16 534位A→G、46位T→G和49位A→C突变, 在癌细胞系和癌患者白细胞mtDNA中分别占50%(3/6)和33.3%(2/6), 健康成人白细胞mtDNA中未见此类型突变;第16 278位C→T突变,在癌细胞系mtDNA中占50%(3/6),显著高于正常人群mtDNA中此位点的多态性变异。表明癌细胞和癌患者白细胞mtDNA重链复制起点及其 相邻D环区的特征性突变可能与细胞癌变/或癌的易感性有关。 Abstract：　To explore the sequence feature of mitochondrial DNA(mtDNA) control region in human carcinoma cells, polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and direct sequence techniques were used to analyze the sequence of mtDNA control region of 6 human carcinoma cell lines versus white blood cells which from 6 tumor patients and 4 normal adults. The T to C mutation at np 16 519, A to G mutation at np 16 534, T to G mutation at np 46, and A to C mutation at np 49 was found in 50% (3/6 cases) of carcinoma cell lines and in 33.3%(2/6 cases) of tumor patients, but it was not found in normal adults. The C to T mutation at np 16 278 was found in 50%(3/6 cases) of carcinoma cell lines, it was significantly higher than that of the polymorphism of normal population. These findings suggest that the typical mutation in the starting area of heavy-strand replication and the first half of D-loop region might probably be associated with carcinogenesis or susceptibility of carcinoma.     

Somatic mutations in the D-loop and decrease in the copy number of mitochondrial DNA in human hepatocellular carcinoma

Somatic mutations in mitochondrial DNA (mtDNA) have been detected in many human cancers, including hepatocellular carcinoma (HCC). The D-loop region was found to be a "hot spot" for mutation in mtDNA of the tumors. However, effects of the D-loop mutations on the copy number of mtDNA in tumor tissues are poorly understood. Using direct sequencing, we examined mutations in the D-loop region of mtDNA in 61 HCCs and the corresponding non-tumor liver tissues. The results revealed that 39.3% of the HCCs carried somatic mutation(s) in the D-loop of mtDNA, and most of these mutations were homoplasmic. Moreover, 37.0% (10/27) of these mutations were T-to-C and G-to-A transitions and 40.7% (11/27) of them were located in the polycytidine stretch between nucleotide position (np) 303 and 309 of mtDNA. In addition, we found that mtDNA copy number of HCC was significantly decreased in 60.5% of the patients with hepatoma, especially in those with somatic mutation(s) in the D-loop of mtDNA (17/24). This decrease in mtDNA copy number was highly associated with the occurrence of point mutations near the replication origin of the heavy-strand of mtDNA. Interestingly, we found that 42.9% (6/14) of the HCCs without mutation in the D-loop had a reduced copy number of mtDNA, indicating that other unidentified factors involved in mitochondrial biogenesis might be defective in the tumor. The results obtained in this study strongly suggest that somatic mutations in the D-loop together with the decrease in the copy number of mtDNA may be an important event during the early phase of liver carcinogenesis.     

How rapidly does the human mitochondrial genome evolve?

The results of an empirical nucleotide-sequencing approach indicate that the evolution of the human mitochondrial noncoding D-loop is both more rapid and more complex than is revealed by standard phylogenetic approaches. The nucleotide sequence of the D-loop region of the mitochondrial genome was determined for 45 members of a large matrilineal Leber hereditary optic neuropathy pedigree. Two germ-line mutations have arisen in members of one branch of the family, thereby leading to triplasmic descendants with three mitochondrial genotypes. Segregation toward the homoplasmic state can occur within a single generation in some of these descendants, a result that suggests rapid fixation of mitochondrial mutations as a result of developmental bottlenecking. However, slow segregation was observed in other offspring, and therefore no single or simple pattern of segregation can be generalized from the available data. Evidence for rare mtDNA recombination within the D-loop was obtained for one family member. In addition to these germ-line mutations, a somatic mutation was found in the D-loop of one family member. When this genealogical approach was applied to the nucleotide sequences of mitochondrial coding regions, the results again indicated a very rapid rate of evolution.     

mtDNA and the origin of Caucasians: identification of ancient Caucasian-specific haplogroups, one of which is prone to a recurrent somatic duplication in the D-loop region.

mtDNA sequence variation was examined in 175 Caucasians from the United States and Canada by PCR amplification and high-resolution restriction-endonuclease analysis. The majority of the Caucasian mtDNAs were subsumed within four mtDNA lineages (haplogroups) defined by mutations that are rarely seen in Africans and Mongoloids. The sequence divergence of these haplogroups indicates that they arose early in Caucasian radiation and gave raise to modern European mtDNAs. Although ancient, none of these haplogroups is old enough to be compatible with a Neanderthal origin, suggesting that Homo sapiens sapiens displaced H. s. neanderthaliensis, rather than mixed with it. The mtDNAs of one of these haplogroups have a unique homoplasmic insertion between nucleotide pair (np) 573 and np 574, within the D-loop control region. This insertion makes these mtDNAs prone to a somatic mutation that duplicates a 270-bp portion of the D-loop region between np 309 and np 572. This finding suggests that certain nonpathogenic mtDNA mutations could predispose individuals to mtDNA rearrangements.     

Mitochondrial DNA variants in a Japanese population of patients with Alzheimer’s disease

The evidence for the role of mitochondria in Alzheimer’s disease (AD) has been well investigated, based on the amyloid hypothesis and its relation to the mitochondrial dysfunction due to oxidative stress. However, contrasting reports describe an unclear picture on the relationship between AD and mitochondrial DNA (mtDNA) variations. Therefore, we analyzed complete mtDNA sequences from 153 AD patients and 129 normal control subjects to determine if inherited mtDNA polymorphisms or rare variants, or both contribute to the etiology of late-onset AD. The results reported herein indicate that inherited mtDNA common polymorphisms could not be the single major causes of AD but that some rare variants in the protein-coding-region may have protective effects for high-risk populations with the APOE e4 allele. Furthermore, our results support the idea that the np956–965 poly-c insertion and 856A>G variant might be a riskfactor for AD.     

线粒体DNA G7444A突变可能影响A1555G突变的表型表达

线粒体12S rRNA和tRNASer(UCN) 基因是导致非综合征型听力损失的两个突变热点区域。作者收集了1个母系遗传感音神经性聋家系, 该家系同时携带线粒体DNA (mtDNA) A1555G和G7444A突变。临床资料分析表明, 该家系包括药物致聋的耳聋外显率(所有耳聋患者/所有母系成员)为58%, 而非药物致聋的耳聋外显率(非药物性聋患者/所有母系成员)为25%, 明显高于其他携带A1555G突变的耳聋家系。先证者的线粒体全序列分析表明, 该线粒体基因组共有28个多态位点, 属于东亚人群B4c1单体型。在这些多态位点中, 除A1555G和G7444A突变外, 未发现其他有功能意义的突变。这表明mtDNA G7444A突变可能加重由A1555G突变造成的线粒体功能缺失, 从而增加耳聋的外显率。     

Analysis of mitochondrial DNA mutations in D-loop region in thyroid lesions

Background

Mitochondrial defects have been associated with various human conditions including cancers.

Methods

We analyzed the mutations at the mitochondrial DNA (mtDNA) in patients with different thyroid lesions. In particular, in order to investigate if the accumulation of mtDNA mutations play a role in tumor progression, we studied the highly variable main control region of mtDNA, the displacement-loop (D-loop) in patients with non-tumor nodular goiters, with benign thyroid adenomas, and with malignant thyroid carcinomas. Total thyroid tumor or goiter samples were obtained from 101 patients, matched with nearby normal tissue and blood from the same subject.

Results

Noticeably, mitochondrial microsatellite instability (mtMSI) was detected in 2 of 19 nodular goiters (10.53%), and 8 of 77 (10.39%) malignant thyroid carcinomas. In addition, 6 patients, including 5 (6.49%) with malignant thyroid carcinomas and 1 (5.26%) with nodular goiter, were found to harbor point mutations. The majority of the mutations detected were heteroplasmic.

General significance

Our results indicate that mtDNA alterations in the D-loop region could happen before tumorigenesis in thyroid, and they might also accumulate during tumorigenesis.     

Nucleotide sequence variation is frequent in the mitochondrial DNA displacement loop region of individual human tumor cells

The mitochondrial DNA (mtDNA) displacement loop (D-loop) regions of 76 various tumor cell lines were examined to investigate the existence of a specific relationship between a somatic mtDNA sequence and initiation and/or progression of a tumor. Based on molecular cloning-sequencing analysis, a nucleotide sequence in the D-loop region in each cell line was found to be homoplasmic. Several site-specific nucleotide variations were found in stomach and liver tumor cell lines more frequently than those in other tumor cell lines. Subsequently, 20 pairs of noncancerous and cancerous parts from stomach and liver tumor tissues were examined. In the liver tumor tissue, 80% of the noncancerous parts exhibited slightly higher heterogeneity than the corresponding cancerous parts. Several site-specific nucleotide variations found in 76 tumor cell lines were also detected in noncancerous or cancerous parts of stomach and liver tumor tissues. However, it remains unclear why the mtDNA D-loop sequence is homoplasmic in each tumor cell line. The data indicate that mtDNA exhibits heterogeneity even in the noncancerous part and a slight decrease in heterogeneity during tumorigenesis and/or tumor progression. Homoplasmy of the mtDNA population in the tumor cell line would be acquired in the cloning process of establishing a cell line. Site-specific nucleotide substitutions might not be directly involved in the tumorigenesis process.     

High frequency of mitochondrial DNA D-loop mutations in Ewing’s sarcoma

Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing’s sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations’ possible involvement in the initiation and/or progression of this rare malignancy.     

Normal variants of human mitochondrial DNA and translation products: the building of a reference data base

Summary A good standard reference for the highly polymorphic human mitochondrial DNA (mtDNA) sequence is essential for studies of normal and disease-related nucleotide variants in the mitochondrial genome. A consensus sequence for the human mitochondrial genome has been derived from thirteen unrelated mtDNA sequences. We report 128 nucleotide variants of the human mtDNA sequence, and 62 amino acid variants of the human mitochondrial translation products, observed in the coding region of these mtDNA sequences.     

Genetic Characteristics of Mitochondrial DNA Was Associated with Colorectal Carcinogenesis and Its Prognosis

Clinical value of mitochondrial DNA has been described in colorectal cancer (CRC). To clarify its role in colorectal carcinogenesis, mitochondrial microsatellite instability (mtMSI) and other markers were investigated in CRCs and their precancerous lesions, as a multitier genetic study. DNA was isolated from paired normal and tumoral tissues in 78 tubular adenomas (TAs), 34 serrated polyps (SPs), and 100 CRCs. mtMSI, nucleus microsatellite instability (nMSI), KRAS mutation, and BRAF mutation were investigated in these tumors and their statistical analysis was performed. mtMSI was found in 30% of CRCs and 21.4% of precancerous lesions. Mitochondrial copy number was higher in SPs than TAs and it was associated with mtMSI in low grade TAs. KRAS and BRAF mutations were mutually exclusive in TAs and SPs. CRCs with mtMSI showed shorter overall survival times than the patients without mtMSI. In CRCs without nMSI or BRAF mutation, mtMSI was a more accurate marker for predicting prognosis. The genetic change of mitochondrial DNA is an early and independent event in colorectal precancerous lesions and mtMSI and mitochondrial contents are associated with the tubular adenoma-carcinoma sequence, resulting in poor prognosis. This result suggested that the genetic change in mitochondrial DNA appears to be a possible prognosis marker in CRC.     

Identification of somatic and germline mitochondrial DNA sequence variants in prostate cancer patients

Prostate cancer (PCa) is the second most frequent cancer among men in the European Union and the most common in the United States. Older age and a positive family history of PCa are important risk factors, but little is known about the disease aetiology. Mitochondria are involved in essential cellular pathways, some of which have been associated with tumorigenesis. We analysed the presence of sequence variants, depletion and rearrangements in the mitochondrial DNA (mtDNA) of PCa patients. Sequencing of the D-loop and genes RNR1 and 2, ND3, ND4L and ND4, and long-range and real-time PCR techniques were carried out on 51 samples (41 from patients and 10 from controls). Normal, hyperplastic or tumour samples were obtained from 17 patients. Six patients' seminal vesicles were also investigated as an additional patient's control tissue (these structures seldom develop tumours). Neither depletion nor mtDNA rearrangements were detected. In contrast, 94 mtDNA sequence variants were identified, 9 previously unreported. The regions presenting more sequence variants were MT-DLOOP (52%), MT-RNR2 (14%) and MT-ND4 (13%). The patients' seminal vesicles studied showed the same set of variants as the corresponding prostate, suggesting either that the pathogenic role of these particular variants is minor or that they participate in the prostatic carcinogenesis in combination with other factors absent in seminal vesicles. Five patients (29.4%) harboured eight somatic changes in the mtDNA. One affects a conserved residue and three have not been previously described. The analysis of other genes in the mtDNA molecule might demonstrate an even higher incidence of mtDNA somatic variants in these PCa patients.     

The MYC dualism in growth and death

Investigations of intraindividual sequence diversity in mtDNA are a key step in exploring the linkage between somatic mutations in mtDNA and mitochondrial genome evolution. This paper reports a directional cloning procedure enabling the isolation of multiple copies of the D-loop region of the mitochondrial genome from the fish Ameiurus nebulosus. Sequence analysis of 708 D-loop molecules revealed eight mutants, an average intraindividual mutation frequency of 1.12%. Three different types of mutations were detected but each derived from a single mutational event. By contrasting the spectrum of nucleotide variation at multiple biological levels, one can investigate the effects of spontaneous mutations on genome evolution. Such hierarchical analysis suggested shifts in the type and distribution of mtDNA (mitochondrial DNA) mutations at different biological levels, indicating the need to recognize three different rates of mtDNA sequence change from the cellular to population level.     

Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA.

The sequences of the displacement-loop (D-loop) regions of mitochondrial DNA (mtDNA) from mouse L cells and human KB cells have been determined and provide physical maps to aid in the identification of sequences involved in the regulation of replication and expression of mammalian mtDNA. Both D-loop regions are bounded by the genes for tRNAPhe and tRNAPro. This region contains the most highly divergent sequences in mtDNA with the exceptions of three small conserved sequence blocks near the 5' ends of D-loop strands, a 225 nucleotide conserved sequence block in the center of the D-loop strand template region, and a short sequence associated with the 3' ends of D-loop strands. A sequence similar to that associated with the 3' termini of D-loop strands overlaps one of the conserved sequence blocks near the 5' ends of D-loop strands. The large, central conserved sequence probably does not code for a protein since no open reading frames are discretely conserved. Numerous symmetric sequences and potential secondary structures exist in these sequences, but none appear to be clearly conserved between species.     

Characterization of mitochondrial genome of Formosan sambar (Rusa unicolor swinhoei)

The complete mitochondrial genome sequence of the Formosan sambar (Rusa unicolor swinhoei) was obtained by DNA sequencing based on PCR fragments amplified by 26 primer pairs designed by ourselves. The results indicated that the mtDNA is 16,505 bp in size. This is the first report on mitochondrial DNA (mtDNA) sequence analysis of the Formosan sambar and the sequence was deposited in the GenBank database under the accession number DQ989636. The complete mitochondrial sequence included the following gene sequences: 12S and 16S rRNAs, 22 tRNAs and 13 protein-coding genes. The base composition of the sequence was as follows: A, 33.51%; T, 28.97%; C, 24.07%; and G, 13.46%. The mitochondrial D-loop region was also analyzed for comparative purposes in the Formosan sambar and 13 other species within the Cervidae family using neighbour-joining method. The phylogenetic tree demonstrated that there are two separate groups, a European type and an Asian type, within the Cervidae family. The D-loop sequences of mtDNA of 24 Formosan sambar animals were compared, and the results showed that the Formosan sambar can be divided into two clades.     

耐力运动员及普通人群线粒体DNA调控区遗传多态性分析

目的：通过ＰＣＲ技术对优秀耐力项目运动员以及普通个体的ｍｔＤＮＡ调控区遗传多态性进行分析,以期发现与运动能力相关联的基因标记。方法：以单根毛发为检材,运用ＰＣＲ技术分析中国优秀耐力性项目运动员（ｎ＝６７）,一般水平运动员（ｎ＝３３）以及普通人群（ｎ＝２０）的线粒体ＤＮＡ调控区（Ｄ－Ｌｏｏｐ）的ＲＦＬＰ。结果：优秀耐力性项目运动员线粒体特定区域ＲＦＬＰ分布与普通人群至显著性差异。结论：一些在优秀运动     

Clinical evaluation and mitochondrial DNA sequence analysis in two Chinese families with aminoglycoside-induced and non-syndromic hearing loss

We report here the clinical, genetic, and molecular characterization of two Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects. Penetrances of hearing loss in BJ105 and BJ106 pedigrees are 67% and 33%, respectively. In particular, three of 10 affected matrilineal relatives of BJ105 pedigree had aminoglycoside-induced hearing loss, while seven affected matrilineal relatives in BJ105 pedigree and six affected matrilineal relatives in BJ106 pedigree did not have a history of exposure to aminoglycosides. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mtDNA variants belonging to haplogroups F3 and M7b. These variants showed no evolutionary conservation, implying that mitochondrial haplotype may not play a significant role in the phenotypic expression of the A1555G mutation in these Chinese pedigrees. However, aminoglycosides and nuclear backgrounds appear to be major modifier factors for the phenotypic manifestation of the A1555G mutation in these Chinese families.