Genomic instability in sporadic colorectal cancer quantitated by inter-simple sequence repeat PCR analysis

Genomic instability plays a major role in cancer by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (inter-SSR) PCR has been developed to provide a rapid and reproducible technique for quantitation of the major type of genomic instability observed in sporadic tumors, namely, that manifesting itself as amplifications, deletions, translocations, and insertions. Evaluation of 59 sporadic colorectal cancers by inter-SSR PCR has demonstrated a wide range of instability, independent of tumor stage at diagnosis. Comparison of these data and the results of microsatellite PCR analysis reveals an association of high genomic instability with loss of heterozygosity but no association with the replication error phenomenon arising from defects in mismatch repair. Genes Chromosom. Cancer 18:19–29, 1997. © 1997 Wiley-Liss, Inc.     

分枝杆菌简单序列重复区间分型方法的研究

目的 构建分枝杆菌简单序列重复区间(inter-simple sequence repeat,ISSR)分型系统,为结核分枝杆菌的基因分型、菌种鉴定以及流行病学的研究提供新的研究工具和研究思路.方法 根据结核分枝杆菌基因组中简单重复序列(SSR)的丰度和频率,设计用于分枝杆菌基因分型的ISSR引物,以分枝杆菌属的DNA为材料,分析DNA浓度、Mg2+浓度、dNTP浓度、Taq酶的含量以及退火温度对ISSR-PCR扩增结果的影响,构建分枝杆菌的ISSR分型体系,并通过该体系筛选获得扩增结果稳定、多态性强的引物.结果 经过优化实验建立了分枝杆菌ISSR-PCR最佳反应体系,20 μl的PCR反应液含有的组分和终浓度分别为:10 ng模板DNA,1 U Taq酶,0.5μmol/L引物,1.5 mmol/LMgCl2,0.2 mmol/L dNTPs.利用该反应体系从10个ISSR引物中,筛选出了3个稳定性高、重复性好的引物,对分枝杆菌属28个菌种的标准菌株及52株结核分枝杆菌临床分离株进行了ISSR遗传多样性分析.结果 显示,28个分枝杆菌菌种,除了结核和牛分枝杆菌之间具有较高的相似性,两两之间存在着高度的遗传多样性;52株结核分枝杆菌菌株在用ISSR进行分型时,大部分菌株表现出了较高的遗传相似性.结论 建立了分枝杆菌属的ISSR分型体系.     

Accumulation of genetic alterations during esophageal carcinogenesis

Using polymerase chain reaction amplification of microsatelliteregions in DNA from 11 epithelial dysplasias of the esophagusand 21 early squamous cell carcinomas, we were able to detectfrequent loss of heterozygosity (LOH) on chromosomes 3p21.3and 9q31 even in low-grade dysplasias. In contrast, we observedfrequent LOHs on chromosomes 9p22 and 17p13 (TP53 locus) onlyin high-grade dysplasias and carcinomas, but not in any low-gradedysplasias. Analysis of LOH at the same four chromosomal regionsIn DNA of five additional minimal carcinomas and accompanyingdysplastic lesions revealed loss of alleles at the loci on 3p21.3and 9q31 throughout various degrees of dysplasia and carcinoma;again, LOHs on 9q22 and 17p13 occurred only in high-grade dysplasiaand carcinoma in situ. Our results indicated that Inactivationof putative tumor suppressor genes on 3p21.3 and 9q31 may beearly genetic events during esophageal carcinogenesis, and thatadditional genetic alterations on 9p22 and 17p13 probably playroles in progression.     

Molecular characterization of Leishamania isolates from China by inter-simple sequence repeat polymerase chain reaction

Leishmania has distinct epidemiological and biological characteristics and causes a variety of clinical symptoms. To understand the genetic diversity and the phylogenetic relationships among Leishmania isolates from China, 29 Leishmania isolates from different geographic origins, vectors, and hosts were analyzed using 21 inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) primers. A total of 864 polymorphic bands were obtained. According to the results of the neighbor-joining phylogenetic tree and principal component analysis, the 29 isolates studied clustered into six groups. Isolates of Leishmania donovani complex from China share the highest similarity with the reference strain of L. donovani (DD8). This study helps to elucidate the genetic relationship among Leishmania isolates from China and similarities between Chinese isolates and World Health Organization reference strains. Furthermore, ISSR-PCR could also be a quick, simple, and reliable method for Leishmania species identification.     

Application of the crypt isolation technique to the assessment of genetic alterations of colorectal carcinomas

The crypt isolation technique (CIT) allows for the isolation of pure tumor crypts from colon tumor tissue. In a previous study we reported on the genetic alterations found in colorectal tumor crypts using the CIT; however, a direct comparison of the genetic alterations found in colorectal carcinomas using either conventional methods (CM) or the CIT has not previously been performed. Here, we analyzed the impact of this method on the genetic analysis of colon tumor cells by comparing the observed frequency of genetic alterations in colon tumors isolated using CM or the CIT. We used a combination of the CIT and the fluorescent polymerase chain reaction assay to accurately assess the incidence of allelic imbalances (AI) at a number of chromosomal loci (17p, 5q, 18q, 1p, 8p, 22q), microsatellite instability (MSI), and mutations of cancer-related genes (p53 and APC genes) in 48 sporadic colorectal carcinomas. In addition, genetic alterations seen in multiploid tumors (defined as tumors with both diploid and aneuploid cell populations) identified by the CIT were also examined. The incidence of AI at the chromosomal loci tested was more frequently detected in samples isolated from tumors using the CIT than in those isolated from the same tumors using CM. In contrast, we observed no differences in the frequency of MSI or cancer-related gene mutation between the two groups. Although there was no difference in the frequency of genetic alterations between tumors with evidence of multiploidy, sorting of diploid and aneuploid populations allowed detection of distinct genetic changes. The crypt isolation method thus appears to be useful in that it allows purification of tumor cells and the accurate assessment of their genetic alterations. In addition, it may also be of benefit in clarifying the genetic profile of multiploid tumor cell populations.     

Application of toxicogenomics to genetic toxicology risk assessment

Based on the assumption that compounds having similar toxic modes of action induce specific gene expression changes, the toxicity of unknown compounds can be predicted after comparison of their molecular fingerprints with those obtained with compounds of known toxicity. These predictive models will therefore rely on the characterization of marker genes. Toxicogenomics (TGX) also provides mechanistic insight into the mode of toxicity, and can therefore be used as an adjunct to the standard battery of genotoxicity tests. Promising results, highlighting the ability of TGX to differentiate genotoxic from non-genotoxic carcinogens, as well as DNA-reactive from non-DNA reactive genotoxins, have been reported. Additional data suggested the possibility of ranking genotoxins according to the nature of their interactions with DNA. This new approach could contribute to the improvement of risk assessment. TGX could be applied as a follow-up testing strategy in case of positive in vitro genotoxicity findings, and could contribute to improve our ability to identify the molecular mechanism of action and to possibly better assess dose-response curves. TGX has been found to be less sensitive than the standard genotoxicity end-points, probably because it measures the whole cell population response, when compared with standard tests designed to detect rare events in a small number of cells. Further validation will be needed (1) to better link the profiles obtained with TGX to the established genotoxicity end-points, (2) to improve the gene annotation tools, and (3) to standardise study design and data analysis and to better evaluate the impact of variability between platforms and bioinformatics approaches.     

Extensive genetic alterations in prostate cancer revealed by dual PCR and FISH analysis

The genetic alterations that underlie prostate tumorigenesis are assumed to comprise gain or loss of specific chromosomal regions, whole chromosomes, or sequence-specific mutations. Existing data have not demonstrated clear specificity of whole chromosome or regional chromosomal gain or loss that characterizes entire individual malignant lesions, or all malignant lesions, within a cancerous prostate. We have analyzed tissues from 13 patients for target sequences by using PCR and FISH techniques on paired malignant or prostatic intraepithelial neoplastic (PIN) and benign samples (usually from different areas of the same paraffin section). We exercised stringent histologic control over these samples by examining small ( < 5 mm²), discrete regions of sectioned benign, malignant, and PIN tissue. The same histologic region was examined on serial sections by FISH and PCR analysis. The tissues were examined for numerical aberrations involving chromosomes 4 (as a control), 7, 8, 10, and the Y by FISH analysis, and for gain or loss of chromosome 7 and chromosomal arms 8p, 10q, and Yp by PCR analysis. The concurrent application of PCR and FISH to microdissected prostatic tissues yielded evidence of higher frequencies of genetic aberration in prostate cancers than those found with either method alone or by other approaches. These results indicate the power of simultaneous genetic assays that are closely linked to specific tumor histology. © 1993 Wiley-Liss, Inc.     

A genetic basis for shared autoimmunity in mouse models

Development of autoimmune disease is the result of activation of the immune system that subsequently leads to tissue destruction. Although the clinical outcome significantly differs between autoimmune diseases, some pathogenic pathways could be shared. During the recent years, intense efforts to find the genetic factors behind development of the complex and polygenic autoimmune diseases have been undertaken. The difficulties in addressing what genetic factors predispose for autoimmunity in humans underline the importance of animal models in the understanding of the general mechanisms behind the initiation of disease. Interestingly, it has been observed in studies of experimental models of autoimmune diseases, that many of the genetic linkages to disease development are located in the same genetic regions and potentially could be controlled by the same gene. Furthermore, comparison of the mouse/rat genetic regions with regions of association to human inflammatory diseases, also demonstrates some homologous loci between species. Some mouse strains can develop disease in more than one model for autoimmunity. This not only argues for some general mechanisms, but it also supports mechanisms related to the specific tissues attacked in the various autoimmune diseases. Here, we will discuss some aspects of shared autoimmunity in mouse models from a genetic point of view.     

Application of MRS to mouse models of neurodegenerative illness

The rapid development of transgenic mouse models of neurodegenerative diseases, in parallel with the rapidly expanding growth of MR techniques for assessing in vivo, non-invasive, neurochemistry, offers the potential to develop novel markers of disease progression and therapy. In this review we discuss the interpretation and utility of MRS for the study of these transgenic mouse and rodent models of neurodegenerative diseases such as Alzheimer's (AD), Huntington's (HD) and Parkinson's disease (PD). MRS studies can provide a wealth of information on various facets of in vivo neurochemistry, including neuronal health, gliosis, osmoregulation, energy metabolism, neuronal-glial cycling, and molecular synthesis rates. These data provide information on the etiology, natural history and therapy of these diseases. Mouse models enable longitudinal studies with useful time frames for evaluation of neuroprotection and therapeutic interventions using many of the potential MRS markers. In addition, the ability to manipulate the genome in these models allows better mechanistic understanding of the roles of the observable neurochemicals, such as N-acetylaspartate, in the brain. The argument is made that use of MRS, combined with correlative histology and other MRI techniques, will enable objective markers with which potential therapies can be followed in a quantitative fashion.     

A genetic basis for shared autoimmunity in mouse models

Development of autoimmune disease is the result of activation of the immune system that subsequently leads to tissue destruction. Although the clinical outcome significantly differs between autoimmune diseases, some pathogenic pathways could be shared. During the recent years, intense efforts to find the genetic factors behind development of the complex and polygenic autoimmune diseases have been undertaken. The difficulties in addressing what genetic factors predispose for autoimmunity in humans underline the importance of animal models in the understanding of the general mechanisms behind the initiation of disease. Interestingly, it has been observed in studies of experimental models of autoimmune diseases, that many of the genetic linkages to disease development are located in the same genetic regions and potentially could be controlled by the same gene. Furthermore, comparison of the mouse/rat genetic regions with regions of association to human inflammatory diseases, also demonstrates some homologous loci between species. Some mouse strains can develop disease in more than one model for autoimmunity. This not only argues for some general mechanisms, but it also supports mechanisms related to the specific tissues attacked in the various autoimmune diseases. Here, we will discuss some aspects of shared autoimmunity in mouse models from a genetic point of view.     

实时定量PCR在强直性肌营养不良1型重复突变检测中的应用

目的 鉴定一个强直性肌营养不良(dystrophy myotonica,DM)家系的致病突变,探讨实时定量PCR能否用于检测导致DM1的CTG重复延展突变.方法 采集家系成员外周血,提取基因组DNA.针对DM1位点DMPK基因内的CTG重复区和DM2位点CNBP基因内的CCTG重复区进行普通PCR、实时荧光定量PCR、微卫星标记连锁分析.结果 CTG重复区普通PCR产物电泳显示患者特有大于2 kb的弥散带.定量PCR显示,患者CTG重复区相对拷贝数约为0.5,CCTG重复区相对拷贝数约为1.在DM1位点的微卫星标记,患者均有共享等位基因;而在DM2位点的大部分标记,患者没有共享等位基因.结论 此DM家系的致病突变是DM1位点的CTG重复延展突变;应用实时定量PCR可以确定高度重复延展片段扩增失败,从而推断重复延展突变的存在,达到快速检测DM1的目的.     

Neurogenesis and alterations of neural stem cells in mouse models of cerebral amyloidosis

The hippocampus in Alzheimer's disease is burdened with amyloid plaques and is one of the few locations where neurogenesis continues throughout adult life. To evaluate the impact of amyloid-beta deposition on neural stem cells, hippocampal neurogenesis was assessed using bromodeoxyuridine incorporation and doublecortin staining in two amyloid precursor protein (APP) transgenic mouse models. In 5-month-old APP23 mice prior to amyloid deposition, neurogenesis showed no robust difference relative to wild-type control mice, but 25-month-old amyloid-depositing APP23 mice showed significant increases in neurogenesis compared to controls. In contrast, 8-month-old amyloid-depositing APPPS1 mice revealed decreases in neurogenesis compared to controls. To study whether alterations in neurogenesis are the result of amyloid-induced changes at the level of neural stem cells, APPPS1 mice were crossed with mice expressing green fluorescence protein (GFP) under a central nervous system-specific nestin promoter. Eight-month-old nestin-GFP x APPPS1 mice exhibited decreases in quiescent nestin-positive astrocyte-like stem cells, while transient amplifying progenitor cells did not change in number. Strikingly, both astrocyte-like and transient-amplifying progenitor cells revealed an aberrant morphologic reaction toward congophilic amyloid-deposits. A similar reaction toward the amyloid was no longer observed in doublecortin-positive immature neurons. Results provide evidence for a disruption of neural stem cell biology in an amyloidogenic environment and support findings that neurogenesis is differently affected among various transgenic mouse models of Alzheimer's disease.     

实时定量PCR在强直性肌营养不良1型重复突变检测中的应用

目的 鉴定一个强直性肌营养不良(dystrophy myotonica,DM)家系的致病突变,探讨实时定量PCR能否用于检测导致DM1的CTG重复延展突变.方法 采集家系成员外周血,提取基因组DNA.针对DM1位点DMPK基因内的CTG重复区和DM2位点CNBP基因内的CCTG重复区进行普通PCR、实时荧光定量PCR、微卫星标记连锁分析.结果 CTG重复区普通PCR产物电泳显示患者特有大于2 kb的弥散带.定量PCR显示,患者CTG重复区相对拷贝数约为0.5,CCTG重复区相对拷贝数约为1.在DM1位点的微卫星标记,患者均有共享等位基因;而在DM2位点的大部分标记,患者没有共享等位基因.结论 此DM家系的致病突变是DM1位点的CTG重复延展突变;应用实时定量PCR可以确定高度重复延展片段扩增失败,从而推断重复延展突变的存在,达到快速检测DM1的目的.     

Detection of inter-spread repeat sequence in genomic DNA sequence

Various types of periodic patterns in nucleotide sequences are known to be very abundant in a genomic DNA sequence, and to play important biological roles such as gene expression, genome structural stabilization, and recombination. We present a new method, named "STEPSTONE", to find a specific periodic pattern of repeat sequence, inter-spread repeat, in which the tandem repeats of the conserved and the not-conserved regions appear periodically. In our method, at first, the data on periods of short repeat sequences found in a target sequence are stored as a hash data, and then are selected by application of an auto-correlation test in time series analysis. Among the statistically selected sequences, the inter-spread repeats are obtained by usual alignment procedures through two steps. To test the performance of our method, we examined the inter-spread repeats in Mycobacterium tuberculosis and Zamia paucijuga genomic sequences. As a result, our method exactly detected the repeats in the two sequences, being useful for identifying systematically the inter-spread repeats in DNA sequence.     

实时定量PCR在强直性肌营养不良1型重复突变检测中的应用

目的 鉴定一个强直性肌营养不良(dystrophy myotonica,DM)家系的致病突变,探讨实时定量PCR能否用于检测导致DM1的CTG重复延展突变.方法 采集家系成员外周血,提取基因组DNA.针对DM1位点DMPK基因内的CTG重复区和DM2位点CNBP基因内的CCTG重复区进行普通PCR、实时荧光定量PCR、微卫星标记连锁分析.结果 CTG重复区普通PCR产物电泳显示患者特有大于2 kb的弥散带.定量PCR显示,患者CTG重复区相对拷贝数约为0.5,CCTG重复区相对拷贝数约为1.在DM1位点的微卫星标记,患者均有共享等位基因;而在DM2位点的大部分标记,患者没有共享等位基因.结论 此DM家系的致病突变是DM1位点的CTG重复延展突变;应用实时定量PCR可以确定高度重复延展片段扩增失败,从而推断重复延展突变的存在,达到快速检测DM1的目的.     

人巨细胞病毒对小鼠宫颈的诱癌作用

用眼科镊将浸透了病毒母液的明胶海绵块塞入小鼠阴道，直抵宫颈的办法，将紫外线灭活的人巨细胞病毒（ＨＣＭＶ）投给昆明种杂交小鼠。约３１周后，有１５０％小鼠诱发了实验性宫颈癌，另一组加用巴豆油促癌剂，小鼠宫颈癌发率为１８８％，对照组癌发率为０。ＨＣＭＶ诱癌组１２例小鼠宫颈癌组织ＨＣＭＶＩＥ（ｉｍｍｅｄｉａｔｅｅａｒｌｙ）抗原均为阳性，血清ＨＣＭＶＩＥ抗体几何平均滴度为１∶１６７２，对照组以上两个指标均为阴性。ＨＣＭＶ诱癌组小鼠外周血淋巴细胞ＡＮＡＥ阳性率为３８９±６８％，对照组为６７８±８０％。     

The relationship of quantitative nuclear morphology to molecular genetic alterations in the adenoma-carcinoma sequence of the large bowel.

The relationship of abnormal nuclear morphology to molecular genetic alterations that are important in colorectal tumorigenesis is unknown. Therefore, Feulgen-stained isolated nuclei from 22 adenomas and 42 carcinomas that had been analyzed for ras gene mutations and allelic deletions on chromosomes 5q, 18q, and 17p were characterized by computerized image analysis. Both nuclear area and the nuclear shape factor representing irregularity correlated with adenoma-carcinoma progression (r = 0.57 and r = 0.52, P < 0.0001), whereas standard nuclear texture, a parameter of chromatin homogeneity, was inversely correlated with progression (r = -0.80, P < 0.0001). The nuclear parameters were strongly interrelated (P < 0.0005). In multivariate analysis, the nuclear parameters were predominantly associated with adenoma-carcinoma progression (P < or = 0.0001) and were not influenced significantly by the individual molecular genetic alterations. Nuclear texture, however, was inversely correlated with fractional allelic loss, a global measure of genetic changes, in carcinomas (r = -0.39, P = 0.011). The findings indicate that nuclear morphology in colorectal neoplasms is strongly related to tumor progression. Nuclear morphology and biologic behavior appear to be influenced by accumulated alterations in cancer-associated genes.     

Comprehensive neurocognitive endophenotyping strategies for mouse models of genetic disorders

There is a need for refinement of the current behavioral phenotyping methods for mouse models of genetic disorders. The current approach is to perform a behavioral screen using standardized tasks to define a broad phenotype of the model. This phenotype is then compared to what is known concerning the disorder being modeled. The weakness inherent in this approach is twofold: First, the tasks that make up these standard behavioral screens do not model specific behaviors associated with a given genetic mutation but rather phenotypes affected in various genetic disorders; secondly, these behavioral tasks are insufficiently sensitive to identify subtle phenotypes. An alternate phenotyping strategy is to determine the core behavioral phenotypes of the genetic disorder being studied and develop behavioral tasks to evaluate specific hypotheses concerning the behavioral consequences of the genetic mutation. This approach emphasizes direct comparisons between the mouse and human that facilitate the development of neurobehavioral biomarkers or quantitative outcome measures for studies of genetic disorders across species.     

Application of quantitative fluorescent PCR with short tandem repeat markers to the study of aneuploidies in spontaneous miscarriages

BACKGROUND: Aneuploidies involve approximately 80% of chromosomal anomalies found in spontaneous miscarriages. Since cytogenetic studies show high rates of failure, we have incorporated the quantitative fluorescent polymerase chain reaction (QF-PCR) technique to the study of numerical chromosome anomalies in miscarriages. METHODS: Multiplex and simple QF-PCR assays have been performed on 160 miscarriage and 34 parental DNA samples analysing specific short tandem repeat (STR) markers for chromosomes 2, 7, 13, 15, 16, 18, 21, 22 and X. Cases successfully karyotyped were used as controls in our study. RESULTS: While maternal contamination could be detected in such cases, a molecular result was obtained for 94% of miscarriages without a cytogenetic one. Thirty-six per cent of them were diagnosed with numerical chromosome anomalies. Parental origin of the extra chromosome and the error stage of meiosis could be also determined. CONCLUSIONS: QF-PCR represents a useful and reliable tool to diagnose aneuploidies in spontaneous miscarriages. It provides information about parental and meiotic origin of anomaly, allowing an appropriate genetic counselling.     

Molecular alterations of E-cadherin gene: possible role in human bladder carcinogenesis

E-cadherin is a transmembrane glycoprotein which mediates a calcium dependent homophilic interaction among epithelial cells. The altered expression and gene mutations of E-cadherin adhesion molecule have been frequently observed in various tumors. Several invasive carcinomas showed cell-cell adhesion loss although the tumor cells expressed considerable amounts of E-cadherin protein. The purpose of this study was to evaluate the role of E-cadherin gene alterations in genesis and progression of bladder carcinoma by mutation analysis of coding region, expression analysis and microsatellite instability at E-cadherin chromosome locus. We analyzed 30 bladder carcinoma (28 transitional and 2 squamous cell carcinoma) at different stage and grade. The mutation analysis showed that in one case there was a presence of a point mutation at codon 846 that consisted of a G (AGC) to C (ACC) transversion resulting in the replacement of R to T. In another sample the sequence analysis revealed a same-sense mutation at the codon 785 (AAC - AAT). The study of E-cadherin mRNA by Northern blot analysis showed that there were no differences of mRNA levels between tumor and normal mucosa samples. We noted that invasive and anaplastic tumors showed a trend to loss of expression, even if we did not find any statistically significant differences. The microsatellite analysis showed the presence of genomic instability in proximity of the E-cadherin gene. Nine out of 30 (30%) specimens presented molecular alterations in at least one out of 2 loci (D16S260 and D16S301) analyzed. The comparison between microsatellite mutations and clinical-histopathological parameters revealed a higher number of alterations in invasive respect to superficial tumors (p=0.014). On the other hand, there were no statistical differences regarding the correlation with pathological grade. These observations, which, nevertheless, need to be confirmed in a larger number of patients, suggest that alterations of E-cadherin gene may be related to pathobiology of bladder cancer development and clinical progression.