Mutation detection with MutH, MutL, and MutS mismatch repair proteins.

Escherichia coli methyl-directed mismatch repair is initiated by MutS-, MutL-, and ATP-dependent activation of MutH endonuclease, which cleaves at d(GATC) sites in the vicinity of a mismatch. This reaction provides an efficient method for detection of mismatches in heteroduplexes produced by hybridization of genetically distinct sequences after PCR amplification. Multiple examples of transition and transversion mutations, as well as one, two, and three nucleotide insertion/deletion mutants, have been detected in PCR heteroduplexes ranging in size from 400 bp to 2.5 kb. Background cleavage of homoduplexes is largely due to polymerase errors that occur during amplification, and the MutHLS reaction provides an estimate of the incidence of mutant sequences that arise during PCR.

Adaptive mutation sequences reproduced by mismatch repair deficiency.

Adaptive reversions of a lac frameshift mutation in Escherichia coli are -1 deletions in small mononucleotide repeats, whereas growth-dependent reversions are heterogeneous. The adaptive mutations resemble instability of simple repeats, which, in hereditary colon cancer, in yeast, and in E. coli occurs in the absence of mismatch repair. The postulate that mismatch repair is disabled transiently during adaptive mutation in E. coli is supported here by the demonstration that the growth-dependent mutation spectrum can be made indistinguishable from adaptive mutations by disallowing mismatch repair during growth. Physiologically induced mismatch repair deficiency could be an important mutagenic mechanism in cancers and in evolution.

Mutations in the MSH3 gene preferentially lead to deletions within tracts of simple repetitive DNA in Saccharomyces cerevisiae.

Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations in the yeast DNA mismatch repair genes MSH2, PMS1, and MLH1 increase the frequency of mutations for normal DNA sequences and destabilize microsatellites. Mutations of human homologs of MSH2, PMS1, and MLH1 also cause microsatellite instability and result in certain types of cancer. We find that a mutation in the yeast gene MSH3 that does not substantially affect the rate of spontaneous mutations at several loci increases microsatellite instability about 40-fold, preferentially causing deletions. We suggest that MSH3 has different substrate specificities than the other mismatch repair proteins and that the human MSH3 homolog (MRP1) may be mutated in some tumors with microsatellite instability.

Mismatch repair SNPs and thyroid cancer susceptibility: a potential role for the MSH6 rs1042821 (Gly39Glu) polymorphism

Abstract of the Poster presented at the 3rd ESPT Conference (European Society of Pharmacogenomics and Personalised Therapy / European Society of Pharmacogenomics and Theranostics), 7-9 October 2015, Budapest.

Characterisation of the oxysterol metabolising enzyme pathway in mismatch repair proficient and deficient colorectal cancer

ACKNOWLEDGMENTS The immunohistochemistry was performed with the support of the Grampian Biorepository. GRANT SUPPORT Rebecca Swan was supported by the Jean Shanks Foundation. This study was supported by funding from Friends of Anchor and the Encompass kick start and SMART:Scotland award schemes of Scottish Enterprise.

Exploiting DNA damage repair defects for effective targeting of acute myeloid leukaemia by PARP inhibitors

Abstract for 24th Biennial Congress of the European Association for Cancer Research, 9–12 July 2016, Manchester, UK. Poster Session: Cancer Genomics, Epigenetics and Genome Instability II: Monday 11 July 2016

Molecular mechanisms of cancer predisposition in HNPCC/Lynch syndrome

Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.

Molecular and clinical characteristics of tricarboxylic acid cycle-associated tumors

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a rare, dominantly inherited tumor predisposition syndrome characterized by benign cutaneous and uterine (ULM) leiomyomas, and sometimes renal cell cancer (RCC). A few cases of uterine leiomyosarcoma (ULMS) have also been reported. Mutations in a nuclear gene encoding fumarate hydratase (FH), an enzyme of the mitochondrial tricarboxylic acid cycle (TCA cycle), underlie HLRCC. As a recessive condition, germline mutations in FH predispose to a neurological defect, FH deficiency (FHD). Hereditary paragangliomatosis (HPGL) is a dominant disorder associated with paragangliomas and pheochromocytomas. Inherited mutations in three genes encoding subunits of succinate dehydrogenase (SDH), also a TCA cycle enzyme, predispose to HPGL. Both FH and SDH seem to act as tumor suppressors. One of the consequences of the TCA cycle defect is abnormal activation of HIF1 pathway ( pseudohypoxia ) in the HLRCC and HPGL tumors. HIF1 drives transcription of genes encoding e.g. angiogenetic factors which can facilitate tumor growth. Recently hypoxia/HIF1 has been suggested to be one of the causes of genetic instability as well. One of the aims of this study was to broaden the clinical definers of HLRCC. To determine the cancer risk and to identify possible novel tumor types associated with FH mutations eight Finnish HLRCC/FHD families were extensively evaluated. The extension of the pedigrees and the Finnish Cancer Registry based tumor search yielded genealogical and cancer data of altogether 868 individuals. The standardized incidence ratio-based comparison of HLRCC/FHD family members with general Finnish population revealed 6.5-fold risk for RCC. Moreover, risk for ULMS was highly increased. However, according to the recent and more stringent diagnosis criteria of ULMS many of the HLRCC uterine tumors previously considered malignant are at present diagnosed as atypical or proliferative ULMs (with a low risk of recurrence). Thus, the formation of ULMS (as presently defined) in HLRCC appears to be uncommon. Though increased incidence was not observed, interestingly the genetic analyses suggested possible association of breast and bladder cancer with loss of FH. Moreover, cancer cases were exceptionally detected in an FHD family. Another clinical finding was the conventional (clear cell) type RCC of a young Spanish HLRCC patient. Conventional RCC is distinct from the types previously observed in this syndrome but according to these results, FH mutation may underlie some of young conventional cancer cases. Secondly, the molecular pathway from defective TCA cycle to tumor formation was intended to clarify. Since HLRCC and HPGL tumors display abnormally activated HIF1, the hypothesis on the link between HIF1/hypoxia and genetic instability was of interest to study in HLRCC and HPGL tumor material. HIF1α (a subunit of HIF1) stabilization was confirmed in the majority of the specimens. However, no repression of MSH2, a protein of DNA mismatch repair system, or microsatellite instability (MSI), an indicator of genetic instability, was observed. Accordingly, increased instability seems not to play a role in the tumorigenesis of pseudohypoxic TCA cycle-deficient tumors. Additionally, to study the putative alternative functions of FH, a recently identified alternative FH transcript (FHv) was characterized. FHv was found to contain instead of exon 1, an alternative exon 1b. Differential subcellular distribution, lack of FH enzyme activity, low mRNA expression compared to FH, and induction by cellular stress suggest FHv to have a role distinct from FH, for example in apoptosis or survival. However, the physiological significance of FHv requires further elucidation.

The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn2+, Mg2+ or Ca2+ ions, unlike human MutL alpha which shows endonuclease activity only in the presence of Mn2+. We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460-658 exhibits Mn2+-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX(2)EX(4)E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and it mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX(2)EX(4)E motif.

Genetic and epigenetic mechanisms of tumor predisposition in hereditary non-polyposis colorectal carcinoma and sporadic cancers

Individuals with inherited deficiency in DNA mismatch repair(MMR) (Lynch syndrome) LS are predisposed to different cancers in a non-random fashion. Endometrial cancer (EC) is the most common extracolonic malignancy in LS. LS represents the best characterized form of hereditary nonpolyposis colorectal carcinoma (HNPCC). Other forms of familial non-polyposis colon cancer exist, including familial colorectal cancer type X (FCCX). This syndrome resembles LS, but MMR gene defects are excluded and the predisposition genes are unknown so far. To address why different organs are differently susceptible to cancer development, we examined molecular similarities and differences in selected cancers whose frequency varies in LS individuals. Tumors that are common (colorectal, endometrial, gastric) and less common (brain, urological) in LS were characterized for MMR protein expression, microsatellite instability (MSI), and by altered DNA methylation. We also studied samples of histologically normal endometrium, endometrial hyperplasia,and cancer for molecular alterations to identify potential markers that could predict malignant transformation in LS and sporadic cases. Our results suggest that brain and kidney tumors follow a different pathway for cancer development than the most common LS related cancers.Our results suggest also that MMR defects are detectable in endometrial tissues from a proportion of LS mutation carriers prior to endometrial cancer development. Traditionally (complex) atypical hyperplasia has been considered critical for progression to malignancy. Our results suggest that complex hyperplasia without atypia is equally important as a precursor lesion of malignancy. Tumor profiles from Egypt were compared with colorectal tumors from Finland to evaluate if there are differences specific to the ethnic origin (East vs.West). Results showed for the first time a distinct genetic and epigenetic signature in the Egyptian CRC marked by high methylation of microsatellite stable tumors associated with advanced stage, and low frequency of Wnt signaling activation, suggesting a novel pathway. DNA samples from FCCX families were studied with genome wide linkage analysis using microsatellite markers. Selected genes from the linked areas were tested for possible mutations that could explain predisposition to a large number of colon adenomas and carcinomas seen in these families. Based on the results from the linkage analysis, a number of areas with tentative linkage were identified in family 20. We narrowed down these areas by additional microsatellite markers to found a mutation in the BMPR1A gene. Sequencing of an additional 17 FCCX families resulted in a BMPR1A mutation frequency of 2/18 families (11%). Clarification of the mechanisms of the differential tumor susceptibility in LS increases the understanding of gene and organ specific targets of MMR deficiency. While it is generally accepted that widespread MMR deficiency and consequent microsatellite instability (MSI) drives tumorigenesis in LS, the timing of molecular alterations is controversial. In particular, it is important to know that alterations may occur several years before cancer formation, at stages that are still histologically regarded as normal. Identification of molecular markers that could predict the risk of malignant transformation may be used to improve surveillance and cancer prevention in genetically predisposed individuals. Significant fractions of families with colorectal and/or endometrial cancer presently lack molecular definition altogether. Our findings expand the phenotypic spectrum of BMPR1A mutations and, for the first time, link FCCX families to the germline mutation of a specific gene. In particular, our observations encourage screening of additional families with FCCX for BMPR1A mutation, which is necessary in obtaining a reliable estimate of the share of BMPR1A-associated cases among all FCCX families worldwide. Clinically, the identification of predisposing mutations enables targeted cancer prevention in proven mutation carriers and thereby reduces cancer morbidity and mortality in the respective families.

The MLH1 c.1852_1853delinsGC (p.K618A) Variant in Colorectal Cancer: Genetic Association Study in 18,723 Individuals

Colorectal cancer is one of the most frequent neoplasms and an important cause of mortality in the developed world. Mendelian syndromes account for about 5% of the total burden of CRC, being Lynch syndrome and familial adenomatous polyposis the most common forms. Lynch syndrome tumors develop mainly as a consequence of defective DNA mismatch repair associated with germline mutations in MLH1, MSH2, MSH6 and PMS2. A significant proportion of variants identified by screening these genes correspond to missense or noncoding changes without a clear pathogenic consequence, and they are designated as "variants of uncertain significance'', being the c.1852_1853delinsGC (p.K618A) variant in the MLH1 gene a clear example. The implication of this variant as a low-penetrance risk variant for CRC was assessed in the present study by performing a case-control study within a large cohort from the COGENT consortium-COST Action BM1206 including 18,723 individuals (8,055 colorectal cancer cases and 10,668 controls) and a case-only genotype-phenotype correlation with several clinical and pathological characteristics restricted to the Epicolon cohort. Our results showed no involvement of this variant as a low-penetrance variant for colorectal cancer genetic susceptibility and no association with any clinical and pathological characteristics including family history for this neoplasm or Lynch syndrome.

基于错配修复系统的基因突变检测生物芯片研究

许多人类疾病和微生物抗药性的产生都是由基因组中单个碱基的替换、插入或缺失等基因突变引起的。因此，迫切需要发展快速、高通量基因突变检测方法来实现对基因疾病和细菌抗药性的早期诊断。本研究针对匕述需求发展了纂于DN八错配修复系统的墓因突变检测生物芯片方法。根据DNA错配修复MtltS蛋白结构与功能上的高度保守性，通过PCR从E.coli K-12基因组中扩一增出DNA错配修复基因，甩石（2.56kb）。通过基因水平的分子操纵，构建了Trx-His6-MutS（THM）、Trx-His6-Linker peptide-Muts（THLM）、Trx-His6-GFP-Linker peptide-MutS（THGLM）和Trx-His6-Linker peptide-Strep-tagll-Linker peptide-MutS（THLSLM）的融合基因并在大肠杆菌中进行了IPTG诱导表达。SDS-PAGE分析表明均有一与预期分子量相应的诱导表达条带出现，其表达量占菌体蛋白的30％左右，且以可溶形式存在。融合蛋白中Trx和His6亲和肤能增加表达蛋白的可溶性及便于蛋白的纯化。连接肤的加入增大了融合蛋白各个成分之间的距离，减少空间位阻，使各个蛋白能够较大程度地保持其原有的生物活性。MLltS融合蛋白的生物活性鉴定结果表明：它们既能识别、结合含有错配碱基的DNA双链，又保留了其它融合成分的生物活性。利用融合蛋白THLSLM中的Strep-tagII与Streptavidin相互作用的天然特性，使融合蛋白THLSLM在StrePtavidin修饰过的芯片基质上自动布阵沉积，制作成蛋白质芯片来识别、结合样品中含有错配或未配刘碱基的DNA双链。THGLM、THLM-Cy3和THLSLM能够使MutS蛋白显示不同的标记信号，通过它们识别并结合固定在DNA芯片基质上的基因片段来发展基因突变检测DNA芯片方法。利用基于MutS的蛋白质芯片和DNA芯片方法对含有不同错配类型、不同长度的DNA片段和错配序列背景对错配结合的影响做了深入研究，证明了MutS介导的基因突变检测生物芯片方法的可行性。基于MutS蛋白的鳌因突变检测生物芯片方法借用了生物系统本身的DNA错配修复（Mismatch Repair，MMR）机制。DNA错配修复过程是许多修复蛋白之间的相互作用共同完成的，其中蛋白MutS、MutL和MutH在肠道细菌例如大肠杆菌的甲基定向错配修复中起决定作用。这些修复蛋白的相关研究也引起了越来越多学者的关注，但对于MutL蛋白的体外生物功能一直存在争议，从而限制了该蛋白的应用研究。本研究利用基因的体外拼接技术构建了融合蛋白Trx-Hi56-Linker peptide-MutL（THLL）、Trx-His6-GFP-Linker peptide-MutL（THGLL）和Trx-His6-Linker peptide-Strep-tagII-Linker peptide-MutL（THLSLL）。非变性凝胶电泳鉴定MutL融合蛋白体外生物功能结果表明：THLL、THGLL和THLsLL都能增加融合蛋白Trx-His6-Linker peptide-MutS（THLM）与含有错配碱基DNA双链的结合，但受ATP浓度变化的影响很大。通过融合蛋白THGLL中绿色荧光蛋白（Green Fluorescent Protein，GFP）的荧光信号或THLSLL中Strep-tagII的特性并利用酶学反应来指示该蛋白的存在，发展了体外研究DNA错配修复蛋白MtuS和MutL之间相互作用的简便方法。本研究以构建的MutS融合蛋白为分子识别元件发展了基因突变检测生物芯片并利用构建的MutL融合蛋白发展了体外研究DNA错配修复蛋白MLuS和MutL之间相互作用的简便方法。建立的融合分子系统方法也为研究其它的蛋白质或生物大分子之间的相互作用提供了一个技术平台。此外，本研究构建的融合蛋白THGLL及其 DNA错配修复蛋白与GFP的融合构想还可用来进行DNA错配修复基因产物的表达与基因突变频率和人类肿瘤恶性程度的相关性研究。

Novel therapeutic targets in colon cancer

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2016

Trypanosoma cruzi MSH2: Functional analyses on different parasite strains provide evidences for a role on the oxidative stress response

Components of the DNA mismatch repair (MMR) pathway are major players in processes known to generate genetic diversity, such as mutagenesis and DNA recombination. Trypanosoma cruzi, the protozoan parasite that causes Chagas disease has a highly heterogeneous population, composed of a pool of strains with distinct characteristics. Studies with a number of molecular markers identified up to six groups in the T. cruzi population, which showed distinct levels of genetic variability. To investigate the molecular basis for such differences, we analyzed the T. cruzi MSH2 gene, which encodes a key component of MMR, and showed the existence of distinct isoforms of this protein. Here we compared cell survival rates after exposure to genotoxic agents and levels of oxidative stress-induced DNA in different parasite strains. Analyses of msh2 mutants in both T. cruzi and T. brucei were also used to investigate the role of Tcmsh2 in the response to various DNA damaging agents. The results suggest that the distinct MSH2 isoforms have differences in their activity. More importantly, they also indicate that, in addition to its role in MMR, TcMSH2 acts in the parasite response to oxidative stress through a novel mitochondrial function that may be conserved in T. brucei. (C) 2010 Elsevier B.V. All rights reserved.