Development of a yeast-based assay system for monitoring microsatellite instability

Simple sequence repeats (microsatellites) are found in all eukaryotic genomes. Instabilities within these sequences have been associated with several human disorders including Huntington's chorea and myotonic dystrophy. Further studies have identified links between microsatellite instability, faulty mismatch repair and certain human cancers, in particular a form of hereditary colorectal cancer. The assay system described here consists of a congenic set of yeast strains mutated in DNA replication and mismatch repair genes and assay plasmids with which it is possible to measure differences in microsatellite stability in the range of 5-850-fold. The development of this technology will allow monitoring of environmental and dietary influences on the genomic stability in the context of human disease.     

Distinct molecular features of colorectal cancer in Ghana

Objectives: While colorectal cancer (CRC) is common, its incidence significantly varies around the globe. The incidence of CRC in West Africa is relatively low, but it has a distinctive clinical pattern and its molecular characteristics have not been studied. This study is one of the first attempts to analyze molecular, genetic, and pathological characteristics of colorectal cancer in Ghana. Methods: DNA was extracted from microdissected tumor and adjacent normal tissue of 90 paraffin blocks of CRC cases (1997–2007) collected at the University of Ghana. Microsatellite instability (MSI) was determined using fragment analysis of ten microsatellite markers. We analyzed expression of mismatch repair (MMR) proteins by immunohistochemistry and sequenced exons 2 and 3 of KRAS and exon 15 of BRAF. Results: MSI analysis showed 41% (29/70) MSI-High, 20% (14/70) MSI-Low, and 39% (27/70) microsatellite-stable (MSS) tumors. Sequencing of KRAS exons 2 and 3 identified activating mutations in 32% (24/75) of tumors, and sequencing of BRAF exon 15, the location of the common activating mutation (V600), did not show mutations at codons 599 and 600 in 88 tumors. Conclusions: Our study found a high frequency of MSI-High colorectal tumors (41%) in Ghana. While the frequency of KRAS mutations is comparable with other populations, absence of BRAF mutations is intriguing and would require further analysis of the molecular epidemiology of CRC in West Africa.     

微卫星不稳定性的生物学意义及其应用前景

微卫星为遍布于人类基因组中的简单重复序列。在人群中,它们呈现高度多态性,并且稳定遗传。微卫星的高度多态性是微卫星不稳定性的表现,它与错配修复基因的缺陷有关。微卫星不稳定性已广泛应用于肿瘤学的研究,并依此提出了肿瘤发生的“增变基因”途径。     

DNA mismatch repair and Lynch syndrome

The evolutionary conserved mismatch repair proteins correct a wide range of DNA replication errors. Their importance as guardians of genetic integrity is reflected by the tremendous decrease of replication fidelity (two to three orders of magnitude) conferred by their loss. Germline mutations in mismatch repair genes, predominantly MSH2 and MLH1, have been found to underlie the Lynch syndrome (also called hereditary non-polyposis colorectal cancer, HNPCC), a hereditary predisposition for cancer. Lynch syndrome affects predominantly the colon and accounts for 2–5% of all colon cancer cases. During more than 30 years of biochemical, crystallographic and clinical research, deep insight has been achieved in the function of mismatch repair and the diseases that are associated with its loss. We review the biochemistry of mismatch repair and also introduce the clinical, diagnostic and genetic aspects of Lynch syndrome.     

转基因小鼠和基因突变小鼠微卫星不稳定性分析

目的探讨微卫星在转基因和基因突变小鼠中的变化,为基因修饰和遗传突变动物的遗传检测和表型分析提供理论依据和技术手段。方法根据文献报道,从GenBank中选取198个等位基因数量多、富含多态性的微卫星位点,以野生型动物为对照,对6种近交系遗传背景的转基因小鼠和5种自然基因突变的近交系小鼠进行微卫星多态性检测,选用1.5%琼脂糖凝胶电泳和STR扫描技术,比较分析微卫星不稳定性。结果共有40个微卫星位点在转基因和基因突变小鼠中表现出多态性。在基因突变小鼠中,微卫星不稳定性有55.6%(10/18)是由纯合变为杂合(Ⅰ型),有3个位点(16.6%,3/18)是纯合突变(Ⅱ型),有5个位点同时存在2种类型的突变。但是在转基因动物中,大多数的微卫星多态性为Ⅰ型突变(87.5%,28/32),只有2个位点(6.2%,2/32)是Ⅱ型突变。另外有2个位点同时存在2种类型的突变。结论基因修饰或基因突变可引起小鼠相关微卫星发生不稳定性,而且某些微卫星位点对基因改变敏感性较高。     

Visualization of mismatch repair complexes using fluorescence microscopy

DNA mismatch repair (MMR) is a surveillance mechanism present in most living organisms, which repairs errors introduced by DNA polymerases. Importantly, loss of MMR function due to inactivating mutations and/or epigenetic silencing results in the accumulation of mutations and as consequence increased cancer susceptibility, as observed in Lynch syndrome patients.During the past decades important progress has been made in the MMR field resulting in the identification and characterization of essential MMR components, culminating in the in vitro reconstitution of 5′ and 3′ nick-directed MMR. However, several mechanistic aspects of the MMR reaction remain not fully understood, therefore alternative approaches and further investigations are needed.Recently, the use of imaging techniques and, more specifically, visualization of MMR components in living cells, has broadened our mechanistic understanding of the repair reaction providing more detailed information about the spatio-temporal organization of MMR in vivo. In this review we would like to comment on mechanistic aspects of the MMR reaction in light of these and other recent findings. Moreover, we will discuss the current limitations and provide future perspectives regarding imaging of mismatch repair components in diverse organisms.     

IRX5 prompts genomic instability in colorectal cancer cells

The Iroquois homeobox gene 5 (IRX5), one of the members of the Iroquois homeobox family, has been identified to correlate with worse prognosis in many cancers, including colorectal cancer (CRC). In this study, upregulation of IRX5 revealed a great reduction in the proliferation of CRC colorectal cancer cell line SW480 and DLD-1, which was accompanied by G1/S arrest, increased expression in cyclin E1, P21, and P53 and a decrease in cyclin A2, B1, and D1. Furthermore, IRX5-mediated an increase expression of RH2A protein, the biomarker of DNA damage. Consequently, the SA-β-gal level is higher in IRX5-overexpression cells compared to control ones, which showed elevated DNA damage triggered cellular senescence. Recapitulating the above findings, IRX5 exhibited higher levels of genomic instability. IRX5 may be a perspective target for cancer therapy and it deserves further investigation.     

Association between polymorphisms of biotransformation and DNA-repair genes and risk of colorectal cancer in Taiwan

The relationship between diet and colorectal cancer has been previously demonstrated and supported with strong epidemiological evidence. The role of genetic polymorphisms has, however, been less well elaborated upon. We conducted a hospital-based case–control study including 727 cases and 736 healthy controls to evaluate the associations of the polymorphic phase-I and -II biotransformations (CYP1A1, CYP1A2, GSTM1, GSTT1, GSTP1, NAT1 and NAT2) and DNA-repair enzymes (XRCC1, XRCC3 and XPD) with the risk of contracting colorectal cancer. We found that men featuring the CYP1A1*2C G/G genotype, the GSTT1 null genotype and XPD 751 with the Gln allele were associated with an elevated risk of colorectal cancer than were men who did not exhibit such genetic features. Multivariate logistic regression analysis revealed that individuals featuring more than two high-risk genotypes increased the colorectal-cancer risk 3.1-fold (OR = 3.1, 95% CI = 1.8–5.2). For women, subjects featuring the CYP1A1*2C G/G genotype and the XRCC3 Thr/Thr genotype faced a 3.1-fold greater risk (95% CI = 1.3–7.0) of colorectal cancer when compared to those featuring the CYP1A1*2C A allele and the XRCC3 Met allele. Taken together, this study suggests that polymorphisms of genes involved in biotransformation and DNA repair could modulate colorectal-cancer risk in Taiwan.     

Genetic instability and darwinian selection in tumours

Genetic instability has long been hypothesized to be a cardinal feature of cancer. Recent work has strengthened the proposal that mutational alterations conferring instability occur early during tumour formation. The ensuing genetic instability drives tumour progression by generating mutations in oncogenes and tumour-suppressor genes. These mutant genes provide cancer cells with a selective growth advantage, thereby leading to the clonal outgrowth of a tumour. Here, we discuss the role of genetic instability in tumour formation and outline future work necessary to substantiate the genetic instability hypothesis.     

Genetic instability and darwinian selection in tumours

Genetic instability has long been hypothesized to be a cardinal feature of cancer. Recent work has strengthened the proposal that mutational alterations conferring instability occur early during tumour formation. The ensuing genetic instability drives tumour progression by generating mutations in oncogenes and tumour-suppressor genes. These mutant genes provide cancer cells with a selective growth advantage, thereby leading to the clonal outgrowth of a tumour. Here, we discuss the role of genetic instability in tumour formation and outline future work necessary to substantiate the genetic instability hypothesis.     

Mouse models of DNA mismatch repair in cancer research

Germline mutations in DNA mismatch repair (MMR) genes are the cause of hereditary non-polyposis colorectal cancer/Lynch syndrome (HNPCC/LS) one of the most common cancer predisposition syndromes, and defects in MMR are also prevalent in sporadic colorectal cancers. In the past, the generation and analysis of mouse lines with knockout mutations in all of the known MMR genes has provided insight into how loss of individual MMR genes affects genome stability and contributes to cancer susceptibility. These studies also revealed essential functions for some of the MMR genes in B cell maturation and fertility. In this review, we will provide a brief overview of the cancer predisposition phenotypes of recently developed mouse models with targeted mutations in MutS and MutL homologs (Msh and Mlh, respectively) and their utility as preclinical models. The focus will be on mouse lines with conditional MMR mutations that have allowed more accurate modeling of human cancer syndromes in mice and that together with new technologies in gene targeting, hold great promise for the analysis of MMR-deficient intestinal tumors and other cancers which will drive the development of preventive and therapeutic treatment strategies.     

Accuracy of MSI testing in predicting germline mutations of MSH2 and MLH1: a case study in Bayesian meta-analysis of diagnostic tests without a gold standard

Microsatellite instability (MSI) testing is a common screening procedure used to identify families that may harbor mutations of a mismatch repair (MMR) gene and therefore may be at high risk for hereditary colorectal cancer. A reliable estimate of sensitivity and specificity of MSI for detecting germline mutations of MMR genes is critical in genetic counseling and colorectal cancer prevention. Several studies published results of both MSI and mutation analysis on the same subjects. In this article we perform a meta-analysis of these studies and obtain estimates that can be directly used in counseling and screening. In particular, we estimate the sensitivity of MSI for detecting mutations of MSH2 and MLH1 to be 0.81 (0.73-0.89). Statistically, challenges arise from the following: (a) traditional mutation analysis methods used in these studies cannot be considered a gold standard for the identification of mutations; (b) studies are heterogeneous in both the design and the populations considered; and (c) studies may include different patterns of missing data resulting from partial testing of the populations sampled. We address these challenges in the context of a Bayesian meta-analytic implementation of the Hui-Walter design, tailored to account for various forms of incomplete data. Posterior inference is handled via a Gibbs sampler.     

Bracken fern-induced malignant tumors in rats: absence of mutations in p53, H-ras and K-ras and no microsatellite instability

Bracken fern (genus Pteridium) has been shown to induce tumors in domestic and experimental animals. Epidemiological studies have also shown an association between human exposure to bracken toxins and increased risk for the development of upper gastrointestinal tract tumors. Our aim in this study was to investigate possible genomic alterations in bracken fern-induced tumors of experimental animals searching for molecular markers that might be used for human epidemiological studies. Using human colorectal carcinogenesis as a molecular model, we examined eight malignant bracken fern-induced tumors of rats for mutations in the genes associated with the "classic pathway" of colorectal cancer, i.e. p53 and ras, and also in the "mutator pathway" by evaluating microsatellite instability. Exons 5-9 of the p53 gene and exons 1 and 2 of the K-ras and H-ras genes were examined by DNA sequencing and no mutations were found in any of the eight tumors. Amplification of five previously validated microsatellite loci (one with mono-, three with di- and one with tetra-nucleotide repeat motifs) in the malignant tumors and in the surrounding normal tissue did not reveal any instability. The involvement of epigenetic alterations or of mutations in other tumor suppressor genes or oncogenes should be further investigated in the search for human epidemiological markers.     

直结肠癌BUBR1的过度表达与TP53突变及微卫星状态关系提示其过度表达与染色体不稳定表型有关

在有丝分裂过程中BUBR1监视微管与着丝点的结合,是保证染色体均等分离的重要分子机制之一.BUBIB变异家谱研究及其敲除模型的研究表明,BUBR1缺陷与染色体不稳定性及肿瘤的发生直接相关.近来在数种人类肿瘤,对BUBR1蛋白过度表达有所报道.但在直结肠癌,BUBR1的过度表达是否与染色体不稳定性的发生有关目前仍无定论.在人类结直肠癌的遗传不稳定性主要表现为两种类型,染色体不稳定性及微卫星不稳定性,它们提示了两条独立的肿瘤发生路径.一般认为不存在高频度微卫星不稳定性表型的肿瘤通过染色体不稳定途径癌变.P53蛋白通过多种机制对维护遗传稳定性起到重要的作用,TP53基因突变经常与染色体不稳定现象并存.DNA倍体情况也是染色体不稳定研究不可缺少的指标.本研究采用免疫组织化学法检测了一组93例进展期散发结直肠癌BUBR1蛋白的表达情况,直接测序法检测TP53变异.高分辨率荧光标记微卫星不稳定检测技术检测微卫星状态,固相激光扫描细胞仪技术检测DNA倍体情况.我们分析了BUBR1表达与三种反映遗传背景的因子的关系.BUBR1蛋白过度表达在人结直肠癌较为常见.在非高频度微卫星不稳定的结直肠癌,BUBR1蛋白过度表达率明显为高(P＜0.01),在TP53基因突变的病例其过度表达率亦较高(P＜0.05).BUBR1蛋白的过度表达与DNA异倍体无统计学相关,但DNA异倍体病例的BuBRl过度表达有偏高倾向.BuBRl表达情况与常用的临床病理学指标无统计学相关.BuBRl过度表达同微卫星状态及TP53突变的关系明确的提示,在人类散发结直肠癌,BUBR1蛋白过度表达与染色体不稳定状态有关.BUBR1过度表达作为一种常见的分子异常,对于肿瘤的早诊预防提供新的标志物.并可能成为治疗的新靶点.     

Serum selenium level in patients with colorectal cancer

Serum selenium levels were determined by fluorometric procedure in 37 patients of both sexes suffering from colorectal cancer. The diagnosis was verified with histopathological examination during surgical treatment. The values found were 46.8±11.2 μg/L. The control group consisted of 230 healthy persons from the same environment as the group of patients. The values found were 64.2±11.5 μg/L. The results of this study are compared with the results of the other research groups analyzing the level of selenium in colorectal cancer.     

四种错配修复基因蛋白在结直肠癌中的表达及临床意义

目的:分析hMLH1、hMSH2、hMSH6和hPMS2四种错配修复基因蛋白在结直肠癌中的表达及其临床意义。方法:随机选取2013年1月至2015年12月广州医科大学附属第三医院结直肠癌患者标本177例,采用免疫组织化学法检测hMLH1、hMSH2、hMSH6和hPMS2蛋白的表达情况,并分析蛋白表达与临床参数间关系。结果:177例结直肠癌组织中,hMLH1蛋白的缺失率为6.2%(11/177),hMSH2蛋白的缺失率为4.0%(7/177),hMSH6蛋白的缺失率为1.7%(3/177),hPMS2蛋白的缺失率为8.0%(14/177),四者之和占所有结直肠癌病例的19.8%(35/177)。四种错配修复基因蛋白表达缺失均与肿瘤发生部位有关(P0.05),另外,hMLH1及hPMS2蛋白的表达缺失还与肿瘤分化程度相关(P0.05),hMSH6蛋白的表达缺失还与肿瘤浸润深度相关(P0.05);而缺失均与年龄、性别、淋巴结转移和远处转移无关(P0.05)。结论:错配修复蛋白的表达在部分结直肠癌组织中出现缺失现象,且与肿瘤部位及分化程度密切相关。hMLH1、hMSH2、hMSH6和hPMS2四种基因的突变,为临床判断预后及拟定治疗方案提供一个有参考价值的依据。     

High serum riboflavin is associated with the risk of sporadic colorectal cancer

BackgroundExperimental results indicate that riboflavin is involved in tumorigenesis. Data regarding the relationship between riboflavin and colorectal cancer (CRC) are limited, and findings vary between observational studies.DesignThis was a case–control retrospective study.ObjectiveThis study aimed to evaluate the associations between serum riboflavin level and sporadic CRC risk.MethodsIn total, 389 participants were enrolled in this study – including 83 CRC patients without family history and 306 healthy controls – between January 2020 and March 2021 at the Department of Colorectal Surgery and Endoscope Center at Xinhua Hospital, Shanghai Jiao Tong University School of Medicine. Age, sex, body mass index, history of polyps, disease conditions (e.g., diabetes), medications, and eight other vitamins were used as confounding factors. Adjusted smoothing spline plots, subgroup analysis, and multivariate logistic regression analysis were conducted to estimate the relative risk between serum riboflavin levels and sporadic CRC risk. After fully adjusting for the confounding factors, an increased risk of colorectal cancer was suggested for individuals with higher levels of serum riboflavin (OR = 1.08 (1.01, 1.15), p = 0.03) in a dose–response relationship.ConclusionsOur results support the hypothesis that higher levels of riboflavin may play a role in facilitating colorectal carcinogenesis. The finding of high levels of circulating riboflavin in patients with CRC warrants further investigation.     

Assessment of microsatellite instability in colorectal cancer patients from Brazil

The replication error status analysis of DNA, through microsatellite instability detection, has become an indispensable tool for hereditary non-polyposis colorectal cancer screening. This study investigated the microsatellite instability in Brazilian individuals presenting colorectal cancer. In this study, 66 patients were clinically analyzed according to Amsterdam II and Bethesda guidelines. Normal and tumour tissues were collected and analyzed for MSI degree according to molecular markers BAT25, BAT26, BAT40, APC–D5S346, D2S123, and D17S250. Eight patients (12.1%) fulfilled the Amsterdam II guidelines, and 15 (22.7%) met the Bethesda guidelines. BAT25 was the most sensitive marker (86.7%), while BAT26 was the least sensitive (66.7%). The specificity of both markers was 100%, but all of the markers must be used since the contribution of each marker to the sensitivity and specificity of the test is complementary. Proximal tumours were significantly predominant among RER⁺ patients. Conclusions: Patients with a family history of colorectal cancer with the tumour in the proximal colon must be screened to replication error status as early as possible in order to avoid the progression of the disease.