Prognostic Significance of Immunohistochemical MSH2 Expression in Prostate Cancer

PURPOSE: There are numerous prostate cancer-related genes that involve in carcinogenesis and tumor progression. Among the genes, DNA mismatch repair genes recognize and repair misincorporated nucleotides during DNA replication. In this analysis, we evaluated the association of hMSH2 which is one of the mismatch repair genes, with risk of aggressive prostate cancer and prostate cancer recurrence. MATERIALS AND METHODS: Immunohistochemistry was performed in 46 patients who diagnosed prostate cancer and underwent radical prostatectomy between January 2006 and December 2012 at Kyung Hee University Hospital at Gangdong. We evaluated an association between the degree of hMSH2 immunohistochemical staining and various clinical variables including prostate-specific antigen (PSA), Gleason score, pathological stage, and biochemical recurrence. The intensity of immunostaining for hMSH2 was divided into 2 groups: low expression group (immunostaining score < 2) and high expression group (immunostaining score ≥2). RESULTS: Although seminal vesicle invasion was marginally associated with the degree of hMSH2 immunohistochemical staining, PSA, Gleason score, lymph node metastasis, presence of lymphatic, perineural, vascular invasion, and extracapsular extension were not associated with the degree of hMSH2 immunohistochemical staining. Furthermore, the association of biochemical recurrence free survival with hMSH2 expression was not statistically significant. CONCLUSIONS: The hMSH2 expression was marginally associated with risk of aggressive prostate cancer such as seminal vesicle invasion. Further evaluation with a larger number of cases is needed to verify these results.

R659X mutation in the MLH1 gene in hereditary non-polyposis colorectal cancer (HNPCC) in an Indian extended family.

Background & objective: Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome), is a genetically heterogeneous disorder that is believed to account for 2–10 per cent of all the colorectal cancer cases. The disease follows autosomal dominant inheritance pattern with high penetrance (85%) and younger age of onset when compared to patients with sporadic tumours. HNPCC is associated with germ-line mutations in the DNA mismatch repair (MMR) genes namely MLH1, MSH2, MSH6, and PMS2. The present study was aimed at analyzing mismatch repair gene(s) in an extended Indian family satisfying the Amsterdam criteria, and extending the analysis to general population to estimate frequency of the mutations/polymorphisms observed. Methods: A total 12 members of the HNPCC family were studied for genetic investigation. Ethnically matched 250 normal individuals were also included as controls to study the observed mutations/ polymorphisms at population level. Results: The analysis resulted in identification of a 1975C>T mutation in exon 17, resulting in substitution of arginine residue with stop codon at codon 659. 655A>G substitution was also observed, resulting in replacement of isoleucine with valine at codon 219. Similar analysis on 250 ethnically matched control subjects revealed complete absence of R659X mutation, while I219V variant was found in 9.8 per cent of the controls. Interpretation & conclusion: R659X mutation correlates with disease phenotype, and 655A>G locus is highly polymorphic. Our study suggested that R659X substitution was prime cause for the disease phenotype in this family. I219V substitution is a polymorphism having no association with the disease onset or segregation. The family members harbouring this mutation were advised to be under regular medical surveillance.

Expression of cyclooxygenase-2 and its relationship with mismatch repair and microsatellite instability in hereditary nonpolyposis colorectal cancer / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate cyclooxygenase-2 (COX-2) expression and its relationship with mismatch repair (MMR) protein expression and microsatellite instability (MSI) in hereditary nonpolyposis colorectal cancer (HNPCC).</p><p><b>METHODS</b>A total of 28 cases of colorectal adenoma and 14 cases of colorectal carcinoma were collected between July 2003 and July 2007 from 33 HNPCC families. Sporadic colorectal adenoma (n=32) and carcinoma patients (n=24) served as controls. With samples of tumor tissues and normal colonic mucosa collected from the patients, the protein expressions of COX-2 and MMR (hMLH1, hMSH2, and hMSH6) were examined with immunohistochemical assay. Frequency of MSI in five standard MSI loci BAT25, BAT26, D2S123, D5S346, and D17S250 were analyzed by means of polymerase chain reaction.</p><p><b>RESULTS</b>The rate of COX-2 high-expression was 53.6% (15/28) and 42.9% (6/14) in HNPCC adenoma and carcinoma; 62.5% (20/32) and 91.7% (22/24) in sporadic adenoma and carcinoma, respectively. That rate was lower in HNPCC carcinoma than in sporadic carcinoma (Pü0.05). MMR-deletion rate and percentage of high-frequency MSI (MSI-H) in HNPCC carcinoma were higher than those in sporadic colorectal carcinoma [both 71.4% (10/14) vs. 12.5% (3/24), both Pü0.01]. Among the 10 MMR-deficient HNPCC carcinoma patients, COX-2 low-expression was observed in 8 cases (80.0%), while COX-2 high-expression was observed in all of the 4 MMR-positive HNPCC carcinoma cases (Pü0.05). In comparison to MMR positive HNPCC carcinoma, HNPCC adenoma, and sporadic carcinoma, COX-2 expression was significantly lower in corresponding MMR-deficient cases (all Pü0.05). The rates of COX-2 low-expression in HNPCC adenoma, HNPCC carcinoma, and sporadic carcinoma with MSI-H were significantly higher than those in the cases with microsatellite stability (all Pü0.05).</p><p><b>CONCLUSION</b>COX-2 is expressed at a low level in HNPCC carcinoma, different from the high COX-2 expression in sporadic carcinoma.</p>

Clinical value of screening hereditary nonpolyposis colorectal cancer in China with protocol recommended by NCCN guidelines / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of the protocol recommended by NCCN-2007 on the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) in China.</p><p><b>METHODS</b>NCCN protocol consists of identifying HNPCC characteristics according to the revised Bethesda Guidelines,genetic counseling with immunohistochemistry and finally genetic testing. Four hundred and nineteen patients diagnosed as colorectal cancer from January 2002 to February 2006 were selected. The hMLH1 and hMSH2 immunostaining were implemented for 90 patients who fulfilled the revised Bethesda Guidelines, in whom 8 patients fulfilling the Amsterdam II (Criteria were classified as group A and the other 82 patients as group B. The frozen tissues were collected from patients who showed loss of hMLH1 or hMSH2 protein expression, then RNA was extracted, and RT-PCR and cDNA sequencing were adopted to detect the germline mutations of hMLH1 and hMSH2.</p><p><b>RESULTS</b>Tumor tissues from 18 patients showed loss of hMLH1 or hMSH2 protein expression (5 patients in group A and 13 in group B). Finally, 21 patients(8 in group A and 13 in group B showed loss expression of MMR protein) were diagnosed as HNPCC, including 2 cases of hMLH1 and 1 case of hMSH2 mutations. These 3 cases with cDNA mutations did not fulfill the Amsterdam II( Criteria, and were finally diagnosed as HNPCC.</p><p><b>CONCLUSION</b>The protocol recommended by NCCN-2007 offers a useful approach to identify HNPCC patients,and reduces the possibility of missed diagnosis of HNPCC.</p>

Study of germline mutation of hMSH2, hMSH6 and hMLH1 and methylation of hMLH1 in microsatellite instability colorectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To study the characteristics of germline mutations of hMLH1, hMSH2 and hMSH6 and promoter methylation status of MLH1 in patients with MSI colorectal cancer.</p><p><b>METHODS</b>Sequence analysis of germline mutation and promoter methylation of MLH1 in 34 prospective collected patients with MSI colorectal cancer were performed.</p><p><b>RESULTS</b>Nineteen out of 34 patients with MSI colorectal cancer were detected with hypermethylation of MLH1,which accounted for 55.9%. 73.7% MSI-H colorectal cancer cases and 33.3% MSI-L colorectal cancer cases were detected with hypermethylation of MLH1 and the difference was significant. Eight germline mutations were found, including 3 MSH6 mutations and 5 MSH2 mutations.</p><p><b>CONCLUSION</b>There are some different characteristics of the germline mutations of hMLH1, hMSH2 and hMSH6 and promoter methylation of MLH1 in Chinese MSI colorectal patients.</p>

Effects of sample re-sequencing and trimming on the quality and size of assembled consensus sequences

The production of nucleic acid sequences by automatic DNA sequencer machines is always associated with some base-calling errors. In order to produce a high-quality DNA sequence from a molecule of interest, researchers normally sequence the same sample many times. Considering base-calling errors as rare events, re-sequencing the same molecule and assembling the reads produced are frequently thought to be a good way to generate reliable sequences. However, a relevant question on this issue is: how many times the sample needs to be re-sequenced to minimize costs and achieve a high-fidelity sequence? We examined how both the number of re-sequenced reads and PHRED trimming parameters affect the accuracy and size of final consensus sequences. Hundreds of single-pool reaction pUC18 reads were generated and assembled into consensus sequences with CAP3 software. Using local alignment against the published pUC18 cloning vector sequence, the position and number of errors in the consensus were identified and stored in MySQL databases. Stringent PHRED trimming parameters proved to be efficient for the reduction of errors; however, this procedure also decreased consensus size. Moreover, re-sequencing did not have a clear effect on the removal of consensus errors, although it was able to slightly increase consensus.

Study on the germline mutation of MSH6 gene in Chinese hereditary nonpolyposis colorectal cancer pedigrees using PCR based sequencing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect the germline mutation of mismatch repair gene (MSH6) in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds fulfilling different clinical criteria.</p><p><b>METHODS</b>The germline mutations of MSH6 gene were detected by PCR based DNA sequencing in 39 unrelated HNPCC probands fulfilling different clinical criteria in which MSH2 and MLH1 mutations were excluded. The exons with missense mutations were analyzed using PCR sequencing in the germline genomic DNA of 137 healthy persons. The expression of MSH6 protein was detected by Envision immunohistochemistry staining in the tumor tissues of the mutational probands.</p><p><b>RESULTS</b>Six germline mutations of MSH6 gene were detected in 39 probands of Chinese HNPCC kindreds, and the mutations distributed in the exon 4, 6, 9 and 10. Four out of six mutations were missense mutation, one was nonsense mutation and the remaining one was insertion mutation in splice site. The results of sequecing for the exons with above four missense mutations in 137 healthy persons' genomic DNA showed that 5 of 137 persons had the missense mutation of c.3488 A to T at codon 1163 of the 6th exon. The mutational rate was approximately 3.65% (5/137), so the mutation could be a single nucleotide polymorphism (SNP). The remaining missense mutations were not found in any germline genomic DNA of 137 healthy persons. Positive expression of MSH6 protein had been identified in the tumor of the SNP proband while the tumors had negative MSH6 protein expression in the rest probands of germline mutation MSH6 gene. The types of mutations and their potential significance were determined by comparing the following databases: http://www.ncbi.nlm.nih.gov/, http://www.ensembl.org/homo-sapies, and http://www.insight-group.org. Five out of the six mutations had not been reported previously and they were new pathological mutations, the rest one was a new SNP.</p><p><b>CONCLUSION</b>Germline mutations of MSH6 gene may play an important role in Chinese HNPCC kindreds fulfilling different clinical criteria. It is necessary to analyze the germline mutations of MSH6 gene using sequencing to identify HNPCC families in the probands in which MSH2 and MLH1 mutation were excluded.</p>

Detección de mutaciones de los genes hMLH1 y hMSH2 del sistema de reparación de malos apareamientos del ADN en familias colombianas sospechosas de cáncer colorrectal no polipósico hereditario (síndrome de Lynch) / Detection mutations in the DNA mismatch repair genes of hMLH1 and hMSH2 genes in Colombian families with suspicion of hereditary non-polyposis colorectal carcinoma (Lynch syndrome)

Introducción. El cáncer colorrectal es la segunda causa de morbilidad y mortalidad por cáncer en los países desarrollados. En Colombia es la quinta causa de muerte entre los diferentes cánceres. Cerca del 75 por ciento de éstos corresponde a cánceres esporádicos, alrededor del 25 por ciento son familiares, y son claramente hereditarios el 5 por ciento. De éstos, el más importantes es el cáncer colorrectal no polipósico hereditario o síndrome de Lynch. Objetivo. Analizar los dos genes más importantes involucrados en el síndrome de Lynch, el hMLH1 y el hMSH2. Materiales y métodos. En 17 familias colombianas que cumplían con los criterios de Ámsterdam II o las pautas de Bethesda, se analizaron por SSCP los 35 exones de estos dos genes y las variantes electroforéticas se secuenciaron. Resultados. Se detectaron 8 mutaciones de línea germinal en las familias analizadas, 7 en el gen hMLH1 y 1 en hMSH2, y se encontró una tasa de detección de mutaciones del 47 por ciento. Seis de las 8 mutaciones encontradas en este estudio han sido previamente reportadas en la literatura. Un cambio de una base en el sitio donador de empalme en el exón 9 del gen hMLH1 (G>A) (dos familias), un cambio A>G en el codón 755 del exón 17, y un cambio G>A en el exón 18. Se detectaron dos nuevas mutaciones, una en el exón 17, un cambio C>T en el codón 640, y una deleción de TG en el codón 184 del exón 3 del gen hMSH2. También se detectó en dos familias un polimorfismo del intrón 13 del hMLH1. Conclusión. Este es el primer estudio realizado en Colombia que detecta mutaciones en el síndrome de Lynch y pretende establecer un programa integral de manejo y prevención

A simple and rapid modified--new method for SNP typing by fragment length discrepant allele specific PCR / 法医学杂志

OBJECTIVE@#To establish a new method for single nucleotide polymorphism (SNP) typing based on allele specific PCR: fragment length discrepant allele specific PCR (FLDAS-PCR), and study the influence on specific extension by introducing a mismatch at the third or fourth 3'-terminal base of allele specific primers.@*METHODS@#For SNP loci rs759117 and rs760887, two allele specific forward primers, with different length and a mismatch introduced at the third or fourth 3'-terminal base, and a public reverse primer were designed for SNP typing. The genotyping of SNP was determined by the two allele specific fragments different in size after polyacrylamide gel and silver staining.@*RESULTS@#The different homozygote genotypes comprised a single band with different size respectively, and the heterozygote genotypes comprised two bands. Typing results were completely consistent with those by direct sequencing. Non-specific primer extension was decreased remarkably after introducing a mismatch at the third or fourth 3'-terminal base of allele specific primers, and the stringency of PCR reaction was cut down.@*CONCLUSION@#FLDAS-PCR is a simple, rapid and efficient new method for SNP typing. During FLDAS-PCR, specific primers with a mismatch at the third or fourth 3'-terminal base have more power to identify two alleles.

Role of mismatch repair genes [h MLHI, h PMS1, h PMS2, GTBP/h MSH6, h MSH2] in the pathogenesis of hepatocellular carcinoma

DNA mismatch repair [MMR] is an important mechanism involved in maintaining fidelity of genomic DNA. Abnormalities in at least one of five MMR genes are implicated in the development of many cancers and the associated micro satellite instability [MSI]. By using a newly developed multiplex reverse transcription -PCR assay, the expression of the five known MMR [hMLH1, hPMS1, hPMS2, GTBP/hMSH6, hMSH2] were evaluated in 33 human HCC cases as well as 16 cases from the normal distant hepatic tissue samples [NDHT] were also evaluated. Twenty- five of them were associated with HCV infection. This was done in an attempt to determine the role of MMR genes in the development of HCC. The beta actin gene was used as an internal control for RNA degradation and DNA contamination and as well as a reference for quantifying the levels of their transcription. Out of the 33 studied HCC cases, 30 cases [90.9%] showed reduction in the expression of one or more of the 5 studied MMR genes. Reduced expression of hMSH2 was found in [71.9%], hMLH1 [53.3%], GTBP [51.1%], hPMS2 [33.3%] and hPMSI [6%]. Correlation analysis showed a strong significant correlation [P= 0.0069] between reduced expression of hPMS2 and GTBP [P=0.0034] as well as hPMS2 and non-cirrhosis [P=0.0197]. Chi-square analysis showed a significant correlation between reduced expression of hMLHl and grade II. On the other hand, 57.1%, 50%, 20%, 18.8% and 6% of the NDHT showed reduced expression of hMSH2, hMLHI, GTBP, hPMS2 and hPMSI respectively. Multivariate analysis showed significant correlation between HCC and hMSH2 [P= 0.008], hMLH1 [P=0.001] and GTBP [P=0.032], also between hPMS2, GTBP and HCC infected with HCV cases [P< 0.001, 0.002]. It could finally be concluded that reduced expression of hPMS2 is likely associated with growth advantage and stimulates proliferation changes that have encouraged malignant development in non- cirrhotic HCV infected patients via acquisition of more genetic damage and the MMR defects that occur at an early stage of hepatocarcinogenesis

Mutation detection of mismatch repair genes in hereditary nonpolyposis colorectal cancer by denaturing high-performance liquid chromatography / 中华外科杂志

<p><b>OBJECTIVES</b>To establish DHPLC method in detecting mutations of mismatch repair genes, hMLH1 and hMSH2, and to identify germline mutations of hMLH1 and hMSH2 in HNPCC kindreds fulfilling Chinese HNPCC criteria.</p><p><b>METHODS</b>Fourteen peripheral blood DNA samples from 14 unrelated HNPCC probands fulfilling Chinese HNPCC criteria were obtained respectively. PCR amplified 35 exons of two main mismatch repair genes, hMLH1 and hMSH2. DHPLC followed by DNA sequencing was used to detect and confirm mutations.</p><p><b>RESULTS</b>a total of 41 colorectal cancers and 19 extracolonic tumors were developed in 14 HNPCC kindreds, and gastric cancer was the most common extracolonic tumor type. Twelve single nucleotide changes were identified by DHPLC in 14 probands. Among them, three were missense mutations, one was a nonsense mutation. Other single nucleotide changes included five single nucleotide polymorphisms, two intron single nucleotide changes, one synonymous mutation. hMLH1 EXON19 CODON749 TAC-->TAG (Tyr-->X), hMSH2 EXON12 CODON629 CAA-->CGA (Gln-->Arg) and hMSH2 EXON15 CODON839 CAT-->CGT (His-->Arg) were new discovered mutations.</p><p><b>CONCLUSIONS</b>(1) DHPLC was considered to be highly effective, convenient technique with consistent results for the mutation detection of hMLH1 and hMSH2 genes. (2) Valid mutations of hMLH1 and hMSH2 genes were identified in about one-third HNPCC kindreds fulfilling Chinese HNPCC criteria and missense mutation was the most common mutational types in this cohort of families.</p>

High Frequency of Microsatellite Instability in Intestinal-type Gastric Cancer in Korean Patients

BACKGROUND: Although there have been some reports on microsatellite alterations in gastric cancer, findings are inconsistent regarding the associations between histological classification and microsatellite instability (MSI). In the present study, we attempted to determine whether Lauren's histological subtypes are related with MSI status. METHODS: Paraffin-embedded tissue samples from 14 diffuse-type and 14 intestinal-type gastric adenocarcinomas were matched up according to patient gender and age. Mononucleotide markers (BAT25 and BAT26) and dinucleotide markers (D2S123, D5S346, and D17S250) were used for MSI analyses. Microsatellite genotypes were categorized in terms of high MSI incidence (MSI-H, > 30% positive marker) or low MSI incidence (MSI-L, < 30% positive marker). Losses of hMLH1 and hMSH2 protein expression were immunohistochemically studied. RESULTS: MSI-H was observed in 11 cases (78%) of the 14 intestinal-type cases as compared to 3 (21%) of the 14 diffuse-type cases (p=0.007). In MSI-H tumors, 10 cases (71%) showed losses of hMLH1 protein expression, while 2 cases (14%) in MSI-L tumors showed losses of hMLH1 protein expression (p=0.006). CONCLUSION: MSI-H tumors are more frequently found in intestinal-type gastric cancer, which suggests the possibility that there are different pathogenic pathways in gastric carcinogenesis according to histologic type.

The analysis for identifying large DNA fragment aberrations of MSH2 and MLH1 genes from familial colorectal cancer in China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the frequency of large fragment aberrations of MSH2 and MLH1 genes from Chinese colorectal cancer (CRC) patients with family history.</p><p><b>METHODS</b>Sixteen exons of MSH2, nineteen exons of MLH1 and seven DNA sequences from the other genes of the samples were screened and checked by multiplex ligation dependent probe amplification (MLPA). First, the methodology was confirmed by testing the positive and negative control samples. Then, 32 CRC or hereditary nonpolyposis colorectal cancer (HNPCC) patients with family history and 20 cases of sporadic CRC were applied to investigate for the large fragment aberrations of MSH2 and MLH1 genes.</p><p><b>RESULTS</b>The genomic DNA fragment deletions of all positive controls were identified and verified by MLPA. Three cases of 32 familial (hereditary) CRC/HNPCC were detected and identified to be the germline heterozygous deletions of MSH2 gene, of which exons 1-7 were deleted from patient No.3, exon 11 from No.25 and exons 2-6 from No.11. However, no genomic DNA fragment aberration of either MSH2 or MLH1 gene was uncovered from 20 sporadic CRC.</p><p><b>CONCLUSION</b>Large DNA fragment aberrations of MSH2 gene was a frequent cause of Chinese HNPCC and CRC patients with family history, and the identification of those aberrations should be included in the regular genetic analysis for CRC/HNPCC patients.</p>

PCR products with heterozygous mutations containing two types of heteroduplexes / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To confirm that PCR products with heterozygous mutations contain not only wide-type and mutant homoduplexes, but also two types of heteroduplexes.</p><p><b>METHODS</b>An insertion-deletion mutation in the exon 1 of KRT9 gene (497delAinsGGCT), which caused Chinese epidermolytic palmoplantar keratoderma (EPPK) was investigated by polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and denaturing high-performance liquid chromatography(DHPLC).</p><p><b>RESULTS</b>Two heteroduplexes and two homoduplexes in the PCR product from the heterozygous mutation of the exon 1 of KRT9 (497delAinsGGCT) were detected.</p><p><b>CONCLUSION</b>PCR products from KRT9 gene with heterozygous mutations contain two types of heteroduplexes. It is without the need to perform heating and cooling PCR products obtained from heterozygous mutations in advance before the mutation screening steps such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), conformation-sensitive gel electrophoresis (CSGE), DHPLC and heteroduplex analysis (HA), etc.</p>

Detection of methylation of hMSH2 gene promoter region of esophageal cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect methylation in promoter region of hMSH2 gene in esophageal cancer.</p><p><b>METHODS</b>Specimens of cancer and normal tissues freshly removed from 32 cases of esophageal cancer patients without previous radiotherapy, chemotherapy or other treatment were preserved at -80 degrees C within 30 min. Methylation specific PCR (MSP) was used to detect methylation of mismatch repair gene (MMR) hMSH2 in promoter region in esophageal cancer and normal esophageal tissues.</p><p><b>RESULTS</b>The frequencies of methylation of hMSH2 gene in promoter region of cancer and normal esophageal tissues were 32.4% (11/32) and 0/30 (0%), respectively, and significant difference was found between the two groups (P < 0.01). The frequency of methylation in elder patients (> or = 70 years old) was significantly higher than that in younger patients (< 70 years old) (P < 0.05). Methylation was less frequently found in grade I-II (18.2%) than in grade III-IV (70.0%) (P < 0.05).</p><p><b>CONCLUSION</b>Methylation of hMSH2 gene in promoter region is related to patients' age and histopathological grade of the esophageal cancer.</p>

DNA repair in Chromobacterium violaceum

Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage

Application of the age-associated injure in mitochondrial DNA / 法医学杂志

Nowadays, the injury in human mitochondrial DNA (mtDNA) is well known to accumulate in various tissues with age. It's significant to further investigate and then apply it to estimation of the age at parenchymas.

Mutagenicity of CORAGRAF and REKAGRAF in the Ames test

Mutagenicity of CORAGRAF (natural coral) and REKAGRAF (hydroxyapatite) was tested in Ames test with and without an external metabolic activation system (S9). The test revealed no mutagenic activity of both locally produced osseous substitutes.

Methylation of mismatch repair gene (MMR) in primary hepatocellular carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To assess the role of methylated mismatch repair (MMR) genes (hMLH1, hMSH2 and hMSH3) in the carcinogenesis and progression of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Samples of 38 cases of HCC along with their corresponding noncancerous tissues, 2 samples of donated normal tissue and 6 cell lines were collected and subject to the methylation-specific PCR (MSP) to examine promoter methylation status of MLH1, MSH2 and MSH3. Six tumor cell lines were analyzed before and after 5-aza-2'-deoxycytidine treatment. In addition, alterations of mRNA expression of MMRs were investigated by quantitative reverse transcription-PCR.</p><p><b>RESULTS</b>CpG island methylation of hMLH1 and hMSH2 was observed in 13.2% (5 of 38 samples) and 68.4% (26 of 38 samples) respectively in HCC, 2.6% (1 of 38 samples) and 55.3% (21 of 38) respectively in corresponding noncancerous tissues, but not in normal control tissues. Promoter methylation of the hMSH2 gene was present in 83.3% of cell lines tested (5/6), but none were observed for the hMLH1 gene. Promoter methylation of the hMSH3 gene was not identified in any tissue samples or cell lines. After 5-aza-2'-deoxycytidine treatment, hMSH2 methylation was induced or completely reversed, and its mRNA expression was increased in most cell lines.</p><p><b>CONCLUSIONS</b>Our results suggest that promoter hypermethylation of hMLH1 and hMSH2 genes is common in HCC. Particularly, there is a high frequency of methylation of hMSH2 in both cancer and noncancerous tissues, but not in normal control tissue. Therefore, hypermethylation of MMR genes, especially hMSH2, may be involved in the carcinogenesis of HCC and may serve as an early diagnostic marker for HCC. The close correlation between hMSH2 methylation and low expression of its mRNA suggests that hMSH2 methylation is an important pathway in the regulation of gene expression.</p>

The effect of curcumin on mismatch repair (MMR) proteins hMSH2 and hMLH1 after ultraviolet (UV) irradiation on HL-60 cells / 华中科技大学学报（医学）（英德文版）

To understand the expression and effect of mismatch repair genes, hMSH2 and hMLH1, and to investigate anti-leukemic cell proliferation mechanism of curcumin, the levels of both genes were detected by multiple comparative RT-PCR. The protein of hMSH2 was determined by flow cytometry (FCM) and the gene mutation of hMSH2 and hMLH1 were detected by PCR-SSCP and microsatellite instability assay. After UV irradiation, the gene expression of hMSH2 and hMLH1 was not increased and showed no response. This phenomenon was not ascribed to gene mutation, because PCP-SSCP and microsatellite instability assay revealed no abnormal gel-shift band in both genes. After irradiation and addition of curcumin, the expression of hMSH2 mRNA increased and the cellular apoptotic rate also increased at the same time. The difference was statistically significant as compared with groups without addition of curcumin and control groups (P < 0.05). Our results suggested that when MMR system was inhibited by the same agents, curcumin can remove this suppression and switch to cellular apoptosis.