The instability within: problems in current analyses of microsatellite instability

Microsatellite instability is regarded as one of the phenotypes of defective DNA mismatch repair and, consequently, as a marker of high risk for cancer. Despite numerous studies, the reported rates for positive microsatellite instability differ widely in each human malignancy. These discrepancies may relate to problems in the methods used. To establish a methodology for an accurate microsatellite instability analysis, technical requirements for a precise assay and biological conditions required for positive microsatellite instability were discussed. First, to describe microsatellite changes in detail, a sensitive detection system with linear detection characteristics and electrophoresis with standardised migration and minimised migration errors are considered to be necessary. Therefore, systems using fluorescent labelling and laser scanning are recommended. For reproducible polymerase chain reactions, it is essential to control the terminal deoxynucleotidyl transferase activity in Taq polymerase. Second, as a biological condition for positive microsatellite instability, feasible selection and combination of microsatellite markers, mutations in specific DNA mismatch repair genes and existence of monoclonal populations enriched sufficiently in a sample are essential. Finally, one possible diagnostic criterion for positive microsatellite instability is proposed, that is the existence of one of the patterns shown in the panel (see Fig. 6) at one or more loci in a set of more than five microsatellite markers.     

Influence of different routes of anti-tumor immunization: alternative induction of tumor immunity and tumor enhancement.

Chickens and quails were immunized in parallel either i.v. or intramuscularly (i.m.) with lectin column-purified antigens from chick embryo cells that were transformed in vitro by avain sarcoma virus (ASV). After five to six injections, immunity of the animals was tested by challenge with ASV into the wing webs. Whereas tumor growth was inhibited after i.v. immunization with respect to incidence rate and time of tumor appearance, tumor growth was enhanced after i.m. injection. Animals that were injected with normal cell antigens served as controls. Spleen cells from only those animals that were immunized i.v. exerted a cytotoxic effect in vitro against ASV-transformed cells, whereas spleen cells from i.m. injected animals, in contrast, suppressed such cytotoxicity. The search for serum blocking or arming factors suggested that sera from i.m. injected animals block cellular cytotoxicity whereas sera from i.v. immunized animals render normal spleen cells cytotoxic (arming effect). The use of viruses from different subgroups and of antigens from gp85-lacking ASV-transformed cells indicates that immune effects were obtained against tumor cell surface antigens that differ from the antigen that is involved in virus neutralization (s-gp85).     

Colletotrichum sublineolum genetic instability assessed by mutants resistant to chlorate

The fungus Colletotrichum sublineolum, causal agent of sorghum anthracnose, presents high variability, genetic instability and host specialization. The aims of the present work were to investigate the mechanisms involved in the genetic instability in this species. Mutants resistant to chlorate and unable to use nitrate (Nit mutants), were obtained spontaneously, isolated and characterized for complementation pattern, reversion frequency and RAPD profile. The results showed that chlorate-resistant mutants could be divided into six phenotypic classes that probably represented mutations in the structural nitrate reductase locus (nit1), in the structural nitrite reductase locus (nit6 and niiA of Neurospora and Aspergillus, respectively), in the specific regulator locus (nit3), in the main regulator locus (nit2), in loci that codified the cofactor containing molybdenum necessary for nitrate reductase activity (NitM), and one or more genes responsible for nitrate intake (crn). In addition, the genetic control of this metabolism in C. sublineolum seems to be similar to other fungi species such as Aspergillus, Neurospora and Fusarium. The high reversion frequency (10⁻⁴ to 10⁻⁵) presented by nit1 mutants suggests that the instability in evaluated strains could be a result of transposable elements activity. The RAPD analysis enabled confirmation that the Nit mutants have a similar genetic background to original strain, and that polymorphism exists among wild-type strains, nit1 mutants and revertants of C. sublineolum. These are important aspects for the later direction of molecular analysis, where these mutants will be used as a tool to isolate the active transposable elements in the C. sublineolum genome.     

Transgenerational carcinogenesis: induction and transmission of genetic alterations and mechanisms of carcinogenesis

Parental exposure, i.e. germ cell exposure to radiation and chemicals, increased the incidence of tumors and malformations in the offspring, and the germ-line alterations that cause cancer are transmissible to further generations. However, tumor incidences were 100-fold higher than those of ordinary mouse mutations and there were apparent strain differences in the types of induced tumors. In human, higher risk of leukemia is reported in the children of fathers who had been exposed to radionuclides at the nuclear reprocessing plants or to diagnostic doses of radiation. However, these findings in mice and men have not been confirmed in the children of atomic bomb survivors in Hiroshima and Nagasaki. Another important finding was that germ-line exposure was very weakly tumorigenic by itself. However, the transmissible alterations caused persistent hypersensitivity to tumor induction in the offspring, e.g. enhanced by postnatal treatment with tumor promoting/carcinogenic agents. The above results suggest that transmissible alterations might be imprinted in germ cells for the future development of cancer by the postnatal environment. Many gene loci concerning immunological, biochemical and physiological function might be involved, and the cumulative changes in such genes may slightly elevate or enhance tumor incidences, although mutations of tumor suppressor genes such as p53 were also detected in some offspring and genomic instability may modify tumor occurrence in transgenerational manner. In fact, Gene Chip analysis showed suppression and/or over-expression of many functional genes rather than cancer-related genes in the preconceptionally irradiated cancer prone progeny.     

Multilocus microsatellite typing shows three different genetic clusters of Leishmania major in Iran

Ten polymorphic microsatellite markers were used to analyse 25 strains of Leishmania major collected from cutaneous leishmaniasis cases in different endemic areas in Iran. Nine of the markers were polymorphic, revealing 21 different genotypes. The data displayed significant microsatellite polymorphism with rare allelic heterozygosity. Bayesian statistic and distance based analyses identified three genetic clusters among the 25 strains analysed. Cluster I represented mainly strains isolated in the west and south-west of Iran, with the exception of four strains originating from central Iran. Cluster II comprised strains from the central part of Iran, and cluster III included only strains from north Iran. The geographical distribution of L. major in Iran was supported by comparing the microsatellite profiles of the 25 Iranian strains to those of 105 strains collected in 19 Asian and African countries. The Iranian clusters I and II were separated from three previously described populations comprising strains from Africa, the Middle East and Central Asia whereas cluster III grouped together with the Central Asian population. The considerable genetic variability of L. major might be related to the existence of different populations of Phlebotomus papatasi and/or to differences in reservoir host abundance in different parts of Iran.     

Analysis of genetic structure of Jamunapari goats by microsatellite markers

Genetic variation at 23 microsatellite loci, population structure, and genetic bottleneck hypothesis were examined for Jamunapari goat population found in Etawah, Uttar Pradesh, India. Estimates of genetic variability such as effective number of alleles and gene diversities revealed substantial genetic variation frequently displayed by microsatellite markers. Number of alleles observed across the microsatellite loci varied from 2 to 10 with an overall mean of 4.913 ± 1.905. Average polymorphism across the studied loci and expected gene diversity in the population were 1.066 ± 0.510 and 0.528 ± 0.237, respectively. Population was observed to be significantly differentiated into different groups, and showed fairly high level of inbreeding (f = 0.189 ± 0.049) and global heterozygote deficit. Bottleneck analysis indicated the introduction of unique/rare alleles by immigrants.     

Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers

Micropropagated plants from two wild-olive species, Olea maderensis and O. europaea ssp. europaea var. sylvestris were screened for genetic stability. O. maderensis shoots were elongated/multiplied on OMG medium with zeatin (9.12 μM), and rooted on 1/2 OMG with NAA (3.22 μM). O. europaea var. sylvestris shoots were elongated/multiplied on OM medium with zeatin, and rooting was optimal after a hormonal shock (IBA 100 μM) followed by transfer to the same medium without growth regulators. In both species, acclimatization was successful and plants looked normal and morphologically identical to the donor field trees. Genetic variability was assessed at several stages of the micropropagation process using flow cytometry (FCM) and nuclear microsatellites (SSR). No changes in ploidy level were found among micropropagated plants, though small deviations, putatively due to the negative effects of cytosolic compounds on propidium iodide staining, between these and field plants were observed. In SSRs analyses, ten SSR markers were able to distinguish between genotypes. No mutations were found in these tested SSR loci among the donor tree and micropropagated plants, suggesting, for the tested markers, genetic uniformity throughout the process. The FCM and SSR results obtained do not exclude the occurrence of other changes in the nuclear genome but, considering the morphological stability of micropropagated plants, indicate that both protocols are suitable and efficient for large scale, true-to-type micropropagation of these two wild olive species.     

应用微卫星标记分析不同桔小实蝇种群的遗传多样性

为探究不同桔小实蝇Bactrocera dorsalis (Hendel)地理种群的遗传变异、入侵来源和扩散情况,利用13对引物对中国南方10省区、泰国、夏威夷、菲律宾和老挝的30个桔小实蝇种群共180个个体的遗传多样性水平进行了研究。 Popgene32和NTSYS-pc2.10e软件分析结果表明：30个不同桔小实蝇种群的遗传相似度在0.3599~0.9153范围内。种群的Nei氏基因多样性指数平均为0.6464±0.1026,Shannon信息指数I平均为1.2845±0.2632, 提示桔小实蝇种群具有较丰富的遗传多样性。UPGMA聚类分析显示,福建地区和海南地区分别独立一支,广东地区和台湾地区种群聚成一支,而广西、泰国、湖南、云南、老挝、四川、 重庆和贵州地区聚为一大支系。据此提出泰国种群和老挝种群是最早入侵我国的种群,云南地区是最早的入侵地,广西地区可能为又一较早入侵地。     

TP53 mutations in colorectal cancer from Tunisia: relationships with site of tumor origin,microsatellite instability and KRAS mutations

Loss of TP53 function through gene mutation is a critical event in the development and progression of colorectal cancer (CRC). Here we examined 51 primary CRC tumors from Tunisia for mutations in TP53 exons 4–9 using PCR-direct sequencing. TP53 status and mutation site/type were than correlated with nuclear protein accumulation, familial and clinicopathologic variables and data on KRAS mutations and microsatellite instability (MSI-H). The TP53 mutation analysis was possible in the tumor of 47 patients and a deleterious somatic mutation has been detected in 59.6 % of the patients (28/47) including 20 (71.4 %) missense mutations, 7 nonsense mutations (25 %) and 1 (3.6 %) frameshift mutation. 89.3 % (25/28) of the detected mutations were in exons 5–8, whereas 10.7 % (3/28) were in exon 4. Among the 27 non frameshift mutations, 89 % (24/27) were transitions and 11 % (3/27) were transversions. 64.3 % (18/27) of the altered amino acids corresponded to arginine. 74 % (20/27) were G>C to A>T transitions, and more than half (14/27) occur at hotspots codons with CpG sites. TP53 mutations correlated closely with TP53 accumulation (p = 0.0090) and inversely with MSI phenotype (p = 0.0658). A KRAS somatic mutation was identified in 25 % (7/28) of the TP53 mutated tumors. All these mutations were G>A transitions in codon 12 and all the tumors with combined alterations but one were distally located and MSS. In conclusion, frequency and types of TP53 mutations and correlations with TP53 protein accumulation, and MSI were as reported for non-Tunisian patients. However, no significant associations have been detected between TP53 mutations and clinicopathological data in Tunisian patients as previously reported.     

卵巢子宫内膜异位症恶变过程中PTEN突变及微卫星不稳定的作用

目的:探讨卵巢子宫内膜异位症恶变过程中PTEN突变及微卫星不稳定的作用.方法:选取子宫内膜异位症相关性卵巢癌(endometriosis associated ovarian cancer,EAOC)26例、不典型子宫内膜异位症(atypical endometriosis,aEMs)7例、典型子宫内膜异位症(endometriosis,EMT)25例,应用PCR结合单链构象多态性(PCR-SSCP)技术及ABI测序系统对各组中PTEN基因第5和第8外显子突变情况检测,应用PCR-SSCP银染方法对5个微卫星监测点Bat25、Bat26、D2S123、D5S346及D17S250的微卫星不稳定(MSI)进行检测分析.结果:EAOC组、aEMs组及EMT组中PTEN的突变率分别为30.76%,14.29%及8%;MSI发生率分别为46.15%.28.67%及0.EAOC组中PTEN基因突变率和MSI发生率均明显高于aEMs和EMT组(P＜0.05),并且EAOC组中PTEN突变率与MSI发生率呈正相关.(r=0.409,P＜0.05).结论:PTEN突变及MSI可能参与了子宫内膜异位症的恶变过程.     

P53 mutations in colorectal cancer from northern Iran: Relationships with site of tumor origin, microsatellite instability and K-ras mutations

CRC-associated P53 mutations have not been studied extensively in non-Western countries at relatively low CRC risk. We examined, for the first time, 196 paraffin-embedded CRC cases from Northern Iran for mutations in P53 exons 5-8 using PCR-direct sequencing. P53 status and mutation site/type were correlated with nuclear protein accumulation, clinicopathologic variables and data on K-ras mutations and high-level microsatellite instability (MSI-H). We detected 96 P53 mutations in 87 (44.4%) cases and protein accumulation in 84 cases (42.8%). P53 mutations correlated directly with stage and inversely with MSI-H. Distal CRCs were more frequently mutated at major CpG hotspot codons [248 (8/66, 12.1%), 175 (7/66, 10.6%), and 245 (7/66, 10.6%)], while in proximal tumors codon 213, emerged as most frequently mutated (5/28, 17.9% vs. 3/66, 4.5%, P = 0.048). Transitions at CpGs, the most common mutation type, were more frequent in non-mucinous (25% vs. 10.4% in mucinous, P = 0.032), and distal CRC (27% vs. 12.5% in proximal, P = 0.02), and correlated with K-ras transversions. Transitions at non-CpGs, second most common P53 mutation, were more frequent in proximal tumors (15.6% vs. 4.7% in distal, P = 0.01), and correlated with K-ras transitions and MSI-H. Overall frequency and types of mutations and correlations with P53 accumulation, stage and MSI-H were as reported for non-Iranian patients. However P53 mutation site/type and correlations between P53 and K-ras mutation types differed between proximal and distal CRC. The codon 213 P53 mutation that recurred in proximal CRC was previously reported as frequent in esophageal cancer from Northern Iran.     

Effects of ovariectomy on microsatellite instability in rat colon tumors induced by 1,2-dimethylhydrazine

To study the effects of ovariectomy on tumorigenesis and microsatellite instability (MSI) in rat colon tumors induced by 1,2-dimethylhydrazine, to elucidate the association between postmenopausal ovarian hormones depletion and MSI pathway in colorectal tumorigenesis. Forty female Wistar rats were randomly divided into two groups: Ovariectomized (Ovx) group and Sham-ovariectomized (Sham-Ovx) group. All rats were injected intraperitoneally with 1,2-dimethylhydrazine (DMH) (20 mg/kg b.w) once a week for 20 weeks. Ten weeks after the final DMH injection, all the rats were sacrificed to collect tumors. Microsatellite instability of six microsatellite loci was detected using fluorescent PCR followed by fragment analysis on automatic DNA sequencer with GeneScan 3.7 software. The tumor multiplicity in the OVX group was significantly higher than that in the Sham-OVX group (3.6 ± 1.4 vs. 2.4 ± 1.6, P < 0.05). The incidence of MSI-positive tumors in OVX group was higher than that in Sham-OVX group (32.1 vs. 10.8%, P < 0.05).The incidence of tumors showing MSI at multiple loci in OVX group was also higher than that in Sham-OVX group (18.9 vs. 2.7%, P < 0.05). Ovariectomy increased tumor formation and the frequency of MSI in DMH-induced colon tumors. It implied that postmenopausal ovarian hormones depletion might influence colorectal tumorigenesis through MSI pathway.     

Low genetic differentiation among widely separated populations of the pearl oyster Pinctada fucata as revealed by AFLP

Genetic variation within and among five populations of the pearl oyster Pinctada fucata, from China (Daya Bay, Sanya Bay and Beibu Bay), Japan (Mie Prefecture) and Australia (Port Stephens) was studied using AFLP. Three primer pairs generated 184 loci among which 91.8-97.3% is polymorphic. An overall genetic diversity of 0.38 among populations and an average of 0.37 within populations (ranging from 0.35 in Japanese population to 0.39 in Beibu Bay population) were observed. Genetic differentiation among the five populations is low but significant as indicated by pairwise G_ST (0.0079-0.0404). AMOVA further shows that differentiation is significant among the five populations but is not significant at a broader geographical scale, among the three groups of Chinese, Japanese and Australian populations or among the two groups of Australian and north Pacific populations. The low level of genetic differentiation indicated that P. fucata populations in the west Pacific are genetically linked. Among the five populations, the Australian one is more differentiated from the others, based on both pairwise AMOVA and G_ST analyses, and is genetically isolated by distance as indicated by Mantel test. However, genetic differences among the three Chinese populations are not correlated with the geographic distances, suggesting that Hainan Island and Leizhou Peninsula may act as barriers blocking gene flow.