Sequence polymorphisms of mitochondrial D-loop and hepatocellular carcinoma outcome

Accumulation of mutations and single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) might be associated with cancer risk and disease outcome. We investigated the prediction power of D-loop SNPs in hepatocellular carcinoma (HCC) patients. No mutation in these HCC patients has prediction power for post-operational survival, whereas two SNP sites (nucleotides 146 T/C and 150 C/T) were identified by the log-rank test for statistically significant prediction of HCC survival. In an overall multivariate analysis, allele 146 was identified as an independent predictor of HCC outcome. The length of survival of patients with allele 146C was significantly shorter than that of patients with allele 146T (relative risk, 2.781; 95% CI, 1.127–6.859; p = 0.026). The analysis of genetic polymorphisms in the mitochondrial D-loop can help identify patient subgroups at high risk of a poor disease outcome.     

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.     

Mitochondrial DNA polymorphisms are associated with the longevity in the Guangxi Bama population of China

Human longevity is an interesting and complicated subject, with many associated variations, geographic and genetic, including some known mitochondrial variations. The population of the Bama County of Guangxi Province of China is well known for its longevity and serves as a good model for studying a potential molecular mechanism. In this study, a full sequence analysis of mitochondrial DNA (mtDNA) has been done in ten Bama centenarians using direct sequencing. Polymorphisms of the displacement loop (D-loop) region of mtDNA and several serum parameters were analyzed for a total of 313 Bama individuals with ages between 10 and 110 years. The results showed that there were seven mitochondrial variations, A73G, A263G, A2076G, A8860G, G11719A, C14766T, and A15326G, and four haplogroups, M(*), F1, D* and D(4) in 10 Bama centenarians. In the D-loop region of mtDNA, the mt146T occurred at a significantly lower frequency in those is the older age group (90-110 years) than in the middle (80-89 years) and in the younger (10-79 years) groups (P < 0.05). The mt146T also had lower systolic blood pressure and serum markers such as total cholesterol, triglyceride and low density lipoprotein than did mt146C in the older age group (P < 0.05). No significant differences were observed between the mt146C and the mt146T individuals in the middle and the younger groups (P > 0.05). The mt5178C/A polymorphisms did not show any significant differences among the three age-groups (P > 0.05), but different nationalities in the Bama County did show a significant difference in the mt5178C/A polymorphisms (P < 0.05). These results suggest that the mt146T/C polymorphisms in Guangxi Bama individuals may partly account for the Bama longevity whereas the mt5178C/A polymorphisms are strongly associated with the nationalities in the Guangxi Bama population.     

Polymorphic variants of folate metabolizing genes (C677T and A1298C <Emphasis Type="Italic">MTHFR</Emphasis> and C1420T SHMT1 and G1958A <Emphasis Type="Italic">MTHFD</Emphasis>) are not associated with the risk of breast cancer in the West Siberian Region of Russia

Breast cancer is one of the most widely distributed cancers in women. We investigated the role of allele variants in the folate metabolizing genes MTHFR (C677T and A1298C alleles), SHMT1 (C1420T allele), and MTHFD (G1258A allele) as a possible factor in predisposition to breast cancer. We determined allele and genotype frequencies of single nucleotide polymorphisms (SNPs) in the case (850 women with sporadic form of breast cancer) and control (810 healthy women) groups. None of the polymorphisms were significantly associated with breast cancer risk. To increase the statistical power of our study, we conducted a meta-analysis which included published genotype data and the results of our work. The meta-analysis revealed no significant association between the studied SNPs and breast cancer risks either.     

两种竹鸡线粒体DNA的遗传变异

采用PCR和直接测序的方法测定灰胸竹鸡(Bambusicola thoracica)与棕胸竹鸡(B.fytchii)线粒体DNA(mtDNA)控制区1142bp的序列,分析二者间的遗传变异。两种竹鸡间共发现32个变异位点,其中20个转换,12个颠换。灰胸竹鸡mtDNA控制区的碱基含量T34.26%、C25.98%、A24.84%和G14.96%,棕胸竹鸡的分别是T34.47%、C25.60%、A25.03%和G14.93%。t-检验分析显示,两种竹鸡mtDNA控制区的T和C含量差异显著。在系统发生树上,两种竹鸡在系统发生树各聚成一支,支持率达到100%。两种竹鸡间的遗传距离是0.0396。根据分子钟计算,它们大约在200万年前分歧进化。推测它们的物种形成主要受更新世第二次寒冷期的影响。     

滇金丝猴线粒体D-loop 片段SNPs 位点的鉴别及初步分析

本文利用已测序的157 个滇金丝猴控制区(D-loop)片段,通过与参考序列比对,鉴别了线粒体D-loop 片段中的52 个SNP (Single Nucleotide Polymorphisms)位点,定义了30 种滇金丝猴单倍型,排除概率为0. 938。谱系及种群遗传结构分析结果与以前利用D-loop 片段的研究结果相似。同时表明基于粪便样品进行保护遗传学、谱系生物地理学、种群遗传学等研究时,与线粒体标记和微卫星标记相比,SNP 标记可能具有一定的优越性,并建议进一步分析滇金丝猴线粒体D-loop 全序列甚至线粒体全基因组上的SNPs 位点的信息,以促进滇金丝猴保护遗传学等研究的开展。     

Characterization of novel and uncharacterized p53 SNPs in the Chinese population--intron 2 SNP co-segregates with the common codon 72 polymorphism

Multiple single nucleotide polymorphisms (SNPs) have been identified in the tumor suppressor gene p53, though the relevance of many of them is unclear. Some of them are also differentially distributed in various ethnic populations, suggesting selective functionality. We have therefore sequenced all exons and flanking regions of p53 from the Singaporean Chinese population and report here the characterization of some novel and uncharacterized SNPs - four in intron 1 (nucleotide positions 8759/10361/10506/11130), three in intron 3 (11968/11969/11974) and two in the 3'UTR (19168/19514). Allelic frequencies were determined for all these and some known SNPs, and were compared in a limited scale to leukemia and lung cancer patient samples. Intron 2 (11827) and 7 (14181/14201) SNPs were found to have a high minor allele frequency of between 26-47%, in contrast to the lower frequencies found in the US population, but similar in trend to the codon 72 polymorphism (SNP12139) that shows a distribution pattern correlative with latitude. Several of the SNPs were linked, such as those in introns 1, 3 and 7. Most interestingly, we noticed the co-segregation of the intron 2 and the codon 72 SNPs, the latter which has been shown to be expressed in an allele-specific manner, suggesting possible regulatory cross-talk. Association analysis indicated that the T/G alleles in both the co-segregating intron 7 SNPs and a 4tagSNP haplotype was strongly associated increased susceptibility to lung cancer in non-smoker females [OR: 1.97 (1.32, 3.394)]. These data together demonstrate high SNP diversity in p53 gene between different populations, highlighting ethnicity-based differences, and their association with cancer risk.     

Polymorphisms in genes involved in the inflammatory response and interaction with NSAID use or smoking in relation to lung cancer risk in a prospective study

Lung cancer risk was investigated in relation to single nucleotide polymorphisms in genes involved in the inflammatory response. The aim was to see if polymorphisms modifying the inflammatory response are associated with risk of lung cancer and if there were interactions between the same polymorphism and factors, which modify an inflammatory response, such as smoking status, duration, and intensity, and use of NSAID. The functional SNPs IL-1B T-31C, IL6 G-174C, IL8 T-251A, IL10 C-592T, COX2 C8473T, COX2 A-1195G and PPARgamma2 Pro(12)Ala were included. A case-cohort study including 428 lung cancer cases and a sub-cohort of 800 persons was nested within a population-based prospective study of 57,053 individuals. Variant allele carriers of IL-1B T-31C were at increased risk of lung cancer (IRR=1.51, 95% CI=1.08-2.12). There was interaction between the polymorphism COX-2 T8473C and smoking status. Thus, non-smoking variant allele carriers were at 5.75-fold (95% CI=1.25-26.43) higher risk of lung cancer than for homozygous wild type allele carriers. Lung cancer risk was similar for all genotype carriers among past and current smokers. There were, however, very few non-smoking lung cancer cases. There was interaction between IL-1B T-31C, COX-2 A-1195G and PPARgamma2 Pro(12)Ala and NSAID use in relation to lung cancer risk. For the two latter, NSAID use was only associated with a lower cancer risk among homozygous wild type allele carriers. p for interaction was 3 x 10(-6) for COX-2 A-1195G and 9 x 10(-5) for PPARgamma2 Pro(12)Ala. The results suggest that NSAID use may modify risk of lung cancer differently depending on the genotype.     

Lack of association between interleukin-13 gene polymorphisms (−1055 C/T and +2044 G/A) in Iranian patients with lung cancer

Lung cancer is one of the leading causes of death from cancer. Both immune cells and tumor cells play a key role in lung cancer immunity by secretion of cytokines and developing type-2 cell-mediated immune response. IL-13 is an immunoregulatory cytokine affecting tumor immunosurveillance by deviation of immune response from Th1 to Th2. In the present study we sought to determine the association of single nucleotide polymorphisms (SNPs) of IL-13 gene at positions +2044 (G/A) and −1055 (C/T) and lung cancer. One hundred forty one patients and 113 controls were recruited; control group was subdivided into smoker and nonsmoker individuals for serum detection. Genotyping was carried out by PCR-RFLP assay and IL-13 detection by ELISA method. No statistically significant difference was found in the frequency of genotypes, alleles, and haplotypes at positions +2044 (G/A) and −1055 (C/T) of IL-13 gene between lung cancer patients and controls. Serum level of IL-13 was not detectable in both groups. The results of this study reveal that although +2044 (G/A) and −1055 (C/T) SNPs in IL-13 are implicated in some pulmonary processes, they do not confer susceptibility to lung cancer in Iranian population.     

Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A→T), 16062 (T→C), and 16135 (G→A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.     

Association of RET Genetic Polymorphisms and Haplotypes with Papillary Thyroid Carcinoma in the Portuguese Population: A Case-Control Study

Thyroid cancer has a multifactorial aetiology resulting from the interaction of genetic and environmental factors. Several low penetrance susceptibility genes have been identified but their effects often vary between different populations. Somatic point mutations and translocations of the REarranged during Transfection (RET) proto-oncogene are frequently found in thyroid cancer. The aim of this case-control study was to determine the effect of four well known RET single nucleotide polymorphisms (SNPs) on the risk for differentiated thyroid carcinoma. A total of 545 Portuguese patients and 543 controls were genotyped by PCR and restriction enzyme analysis, for the following SNPs: G691S (exon 11, rs1799939 G/A), L769L (exon 13, rs1800861 T/G), S836S (exon 14, rs1800862 C/T), and S904S (exon 15, rs1800863 C/G). The minor allele of S836S was overrepresented in patients with papillary thyroid carcinoma (PTC) when compared to controls (OR 1.57; 95% CI 1.05–2.35; p = 0.026). The GGTC haplotype was also overrepresented in PTC (OR 2.51; 95% CI 1.07–5.91; p = 0.029). No associations were found in follicular thyroid carcinoma (FTC). Multivariate logistic regression analysis showed no differences regarding gender, age at diagnosis, lymph node or distant metastasis. However, a near significant overrepresentation of the minor alleles of G691S and S904S was found in patients with tumours greater than 10 mm of diameter at diagnosis. These data suggest that the RET S836S polymorphism in exon 14 and the GGTC haplotype are risk factors for PTC, but not FTC, and that the G691S/S904S polymorphisms might be associated with tumour behaviour.     

The Complete Mitochondrial Genome Sequence and Characterization of Single-Nucleotide Polymorphisms in the Control Region of the Asian Seabass (Lates calcarifer)

We determined the complete mtDNA nucleotide sequence of Lates calcarifer using the shotgun sequencing method. The mitochondrial DNA (mtDNA) was 16,535 base pairs (bp) in length, and contained 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and one major noncoding control region (CR). The CR was unusually short at only 768 bp. A striking feature of the mitochondrial genome was the high G+C content (46.1%), which is among the highest in fish. The gene order was identical to that of a typical vertebrate. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes of 30 fish species representing 14 suborders clearly showed Lates calcarifer was located in the cluster of fish species from the order Perciformes, supporting the traditional systematic classification. We characterized single-nucleotide polymorphisms (SNPs) in the CR by sequencing the complete CR of 25 individuals obtained from Australia and Singapore. A total of 68 SNPs were detected. Eighteen SNPs were fixed with alternative nucleotides in Australian and Singapore seabass, and these SNPs could be used for differentiating fish from the two countries.     

Polymorphisms in the XPG gene and risk of gastric cancer in Chinese populations

DNA repair genes play an important role in maintaining stability and integrity of genomic DNA. Polymorphisms in nucleotide excision repair genes may cause variations in DNA repair capacity phenotype and thus contribute to cancer risk. In this case-control study of 1,125 gastric cancer cases and 1,196 cancer-free controls, we investigated the association between three functional single nucleotide polymorphisms (SNPs, rs2296147T > C, rs2094258C > T and rs873601G > A) in the xeroderma pigmentosum group G (XPG) gene and gastric cancer risk. We used the Taqman assays to genotype these three SNPs and logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). We found that only the rs873601A variant genotypes were associated with a significant higher risk for gastric adenocarcinoma (adjusted OR = 1.30, 95% CI = 1.03-1.64 for AA vs. GG and adjusted OR = 1.23, 95% CI = 1.01-1.49 for AA vs. GG/AG). Stratification analysis indicated that this risk was more pronounced in subgroups of older age (>59 years), males, ever-smokers, and patients with NGCA. All these were not found for the other two SNPs (rs2296147T > C and rs2094258C > T). We then performed expression analysis using gastric cancer adjacent normal tissues from 141 patients and found that the A variant allele was associated with non-significantly reduced expression of XPG mRNA (P(trend) = 0.107). Further analysis using mRNA expression data from the HapMap suggested that the A allele was associated with significantly reduced expression of XPG mRNA in normal cell lines for 45 Chinese (P(trend) = 0.003) as well as for 261 subjects with different ethnicities (P(trend) = 0.001). These support the hypothesis that functional XPG variants may contribute to the risk of gastric cancer. Larger studies with different ethnic populations are warranted to validate our findings.     

Sequence variations of mitochondrial DNA D-loop region are associated with familial nasopharyngeal carcinoma

Mitochondrial DNA (mtDNA) D-loop has been identified as a frequent hot spot of mutations in various tumors. The aim here was to investigate the sequence variations of mitochondrial D-loop region in familial nasopharyngeal carcinoma (FNPC) and their possible associations with cancer risk. 29 subjects from 4 Chinese NPC families and 20 sporadic NPC as well as 122 cases of normal control were recruited. mtDNA extracted from peripheral blood was examined by PCR-based assay for D-loop sequence variations, followed by sequencing analysis. Compared with normal control, four high variations and 6 unrepoted novel polymorphisms were found. Particularly, the np16362 and 16519T to C variants show significantly higher (100%, 81.8%) and lower (0, 22.7%) frequencies in FNPC and unaffected pedigree members, respectively. The occurrence of mitochondrial microsatellite instability (mtMSI) at D310 in experimental groups was statistically significantly higher than in normal control (53.3%). Likewise, in Base Variation Rate consistent with the result, there was a statistically significant difference compared with NC (6.05%). Our results indicated that mtDNA exhibited a high rate of sequence variants in patients with NPC and pedigree members and the mtDNA np16362, np16519 variants and mtMSI at D310 are associated with an increased risk of familial nasopharyngeal carcinoma in pedigree members from families with NPC, which might be involved in the NPC carcinogenesis.     

Association of ADIPOR2 with liver function tests in type 2 diabetic subjects

Objective: Adiponectin protects against liver dysfunction in insulin‐resistant states such as obesity and type 2 diabetes (T2DM), but the role of adiponectin receptors in this disorder is largely unknown. We studied whether common single‐nucleotide polymorphisms (SNPs) in ADIPOR1 and ADIPOR2 are associated with liver function tests (LFTs) in human subjects with various degrees of insulin resistance. Methods and Procedures: Serum alanine (ALT) and aspartate (AST) aminotransferases, homeostasis model assessment of insulin resistance (HOMA‐IR), ?8503 G/A (rs6666089) and +5843 C/T (rs1342387) SNPs in ADIPOR1, ?64,241 T/G (rs1029629) and +33447 C/T (rs1044471) SNPs in ADIPOR2 were assessed in 700 white subjects from a population‐based study. Results: In nondiabetic subjects, the at‐risk alleles for the common ?64,241 T/G and +33447 C/T SNPs in ADIPOR2 were associated with increased circulating adiponectin (P < 0.05 to P < 0.005), but not with LFT. Conversely, in T2DM subjects (who are at risk for liver dysfunction), the same alleles were associated with increased serum ALT and AST (P < 0.05 to P < 0.0001), but not with circulating adiponectin. No significant associations with these parameters were evident for the common ?8503 G/A and +5843 C/T SNPs in ADIPOR1. In a replication study, the ?64,241 T/G and +33447 C/T SNPs in ADIPOR2 were associated with ALT and AST (P < 0.05 to P < 0.0001) in pooled obese and T2DM subjects. Discussion: Common SNPs in ADIPOR2 are associated with LFT in T2DM subjects, which suggests a possible role of this receptor in liver dysfunction associated with insulin resistance.     

Neighboring-nucleotide effects on the mutation patterns of the rice genome

DNA composition dynamics across genomes of diverse taxonomy is a major subject of genome analyses. DNA composition changes are characteristics of both replication and repair machineries. We investigated 3,611,007 single nucleotide polymorphisms （SNPs） generated by comparing two sequenced rice genomes from distant inbred lines （subspecies）, including those from 242,811 introns and 45,462 protein-coding sequences （CDSs）. Neighboring-nucleotide effects （NNEs） of these SNPs are diverse, depending on structural content-based classifications （genomewide, intronic, and CDS） and sequence context-based categories （A/C, A/G, A/T, C/G, C/T, and G/T substitutions） of the analyzed SNPs. Strong and evident NNEs and nucleotide proportion biases surrounding the analyzed SNPs were observed in 1-3 bp sequences on both sides of an SNP. Strong biases were observed around neighboring nucleotides of protein-coding SNPs, which exhibit a periodicity of three in nucleotide content, constrained by a combined effect of codon-related rules and DNA repair mechanisms. Unlike a previous finding in the human genome, we found negative correlation between GC contents of chromosomes and the magnitude of corresponding bias of nucleotide C at -1 site and G at ＋1 site. These results will further our understanding of the mutation mechanism in rice as well as its evolutionary implications.     

Different mutation patterns of mitochondrial DNA displacement-loop in hepatocellular carcinomas induced by N-nitrosodiethylamine and a choline-deficient l-amino acid-defined diet in rats

Mutations of the mitochondria DNA (mtDNA) displacement loop (D-loop) were investigated to clarify different changes of exogenous and endogenous liver carcinogenesis in rats. We induced hepatocellular carcinomas (HCCs) in rats with N-nitrosodiethylamine (DEN) and a choline-deficient l-amino acid-defined (CDAA) diet. DNAs were extracted from 10 HCCs induced by DEN and 10 HCCs induced by the CDAA diet. To identify mutations in mtDNA D-loop, polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Mutations were detected in 5 out of 10 HCCs (50%) induced by DEN. Four out of 5 mutations were G/C to A/T transitions at positions 15707, 15717, 15930, and 16087, and one T/A to C/G transition at position 15559. By contrast, no mutations were found in 10 HCCs induced by the CDAA diet. These results demonstrated that mutations in mtDNA D-loop occur in rat HCCs induced by DEN but not by the CDAA diet, suggesting that mtDNA D-loop is a target of exogenous liver carcinogenesis in rats.     

Somatic mutations in mitochondrial genome and their potential roles in the progression of human gastric cancer

Background

Somatic mutation in mitochondrial DNA (mtDNA) has been proposed to contribute to initiation and progression of human cancer. In our previous study, high frequency of somatic mutations was found in the D-loop region of mtDNA of gastric cancers. However, it is unclear whether somatic mutations occur in the coding region of mtDNA of gastric cancers.

Methods

Using DNA sequencing, we studied 31 gastric cancer specimens and corresponding non-cancerous stomach tissues. Moreover, a human gastric cancer SC-M1 cell line was treated with oligomycin to induce mitochondrial dysfunction. Cisplatin sensitivity and cell migration were analyzed.

Results

We identified eight somatic mutations in the coding region of mtDNAs of seven gastric cancer samples (7/31, 22.6%). Patients with somatic mutations in the entire mtDNA of gastric cancers did not show significant association with their clinicopathologic features. Among the eight somatic mutations, five point mutations (G3697A, G4996A, G9986A, C12405T and T13015C) are homoplasmic and three mutations (5895delC, 7472insC and 12418insA) are heteroplasmic. Four (4/8, 50%) of these somatic mutations result in amino acid substitutions in the highly conserved regions of mtDNA, which potentially lead to mitochondrial dysfunction. In addition, in vitro experiments in SC-M1 cells revealed that oligomycin-induced mitochondrial dysfunction promoted resistance to cisplatin and enhanced cell migration. N-acetyl cysteine was effective in the prevention of the oligomycin-enhanced migration, which suggests that reactive oxygen species generated by defective mitochondria may be involved in the enhanced migration of SC-M1 cells.

General Significance

Our results suggest that somatic mtDNA mutations and mitochondrial dysfunction may play an important role in the malignant progression of gastric cancer.