Association between methylation in mismatch repair genes,V600E BRAF mutation and microsatellite instability in colorectal cancer patients

Colorectal cancer (CRC) corresponds to the third most prevalent type of cancer. Its origins can either be sporadic or inherited, being Lynch syndrome the most common form of hereditary CRC. The activation of BRAF oncogene, inactivation of mismatch repair genes by methylation of CpG islands, and microsatellite instability (MSI) have been reported to be involved in CRC development. The goal of the study was to characterize CRC tumors using clinical and molecular criteria through association and cluster analysis. Amsterdam II and Bethesda guidelines and molecular variables were analyzed in 77 patients from Brazil. The replication error (RER) status, based in microsatellite instability, showed association with metachronous tumor, MLH1 gene methylation and inverse association with left-sided and synchronous tumors. The PMS2 gene was considered the best predictor for differentiating levels of methylation and the mononucleotide were considered the best markers to evaluate RER status. The cluster 1 was characterized of individuals over 60 years of age, female, right-sided tumor, high microsatellite instability, and metachronous or synchronous tumors. The individuals in cluster 2 were younger than 45 years of age, male and showed left sided or rectum tumors, and microsatellite stability. Even though it was not observed a significant association, a higher number of individuals with family history of cancer and tumors without promoter methylation were found in cluster 2. The V600E mutation did not show association with clinical or molecular characteristics. Evaluation of MSI and methylation of MLH1 and PMS2 genes should be considered in order to assist with clinical diagnosis.     

Microsatellite instability typing in serum and tissue of patients with colorectal cancer: comparing real time PCR with hybridization probe and high-performance liquid chromatography

Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI⁺). MSI is detected in about 15 % of all CRCs; 3 % are of these are associated with Lynch syndrome and the other 12 % are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI⁺ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100 % in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.     

Analysis of genome instability in breast cancer

Breast cancer is a heterogeneous disease, previously associated with genomic instability. Our aim was to analyze microsatellite markers in order to determine patterns and levels of instability, as well as possible correlations with histopathological parameters. Polymerase chain reaction was used to characterize microsatellite instability (MSI) and loss of heterozygosity (LOH) in 107 breast carcinomas at twelve microsatellite loci. Some of the markers were selected because of their relation to steroid hormone metabolism, which seems to be related to sporadic breast cancer risk. D5S346 and D17S250 markers showed a statistically significant frequency of MSI. LOH in D3S1611, D17S250, AR and ER-β were associated with some parameters of worse prognosis. Marker group analysis showed that CYP19, AR and ER-β were related to histological grade III, ER-negative and PR-negative cases. Our results suggest that marker group analysis may be preferred to the single marker strategy, being predictive of worst prognosis when single markers are unable to provide such information. A further evaluation of steroid metabolism genes and their association with low penetrance genes in breast cancer may be useful.     

结直肠腺瘤中的微卫星不稳定性类型

应用微切割 聚合酶链反应 单链长度多态性 (PCR SSLP)的方法 ,检测 1 6个微卫星位点在 5 9例 6 2个结直肠腺瘤标本的微卫星不稳定性状态 .结果表明 :腺瘤 1 6个位点的总微卫星不稳定性(microsatelliteinstability ,MSI)发生率为 1 4 4 % ,MSI H所占的比率为 9 7% ;在 1 0例可以同时微切割得到腺瘤和癌变成分的病例中 ,腺瘤和癌变成分在每个微卫星位点的改变情况不完全相同 ,并且当在某一位点同时表现为阳性时 ,部分凝胶电泳的图像相同 ,而部分不同 ;在某些位点表现为癌变成分的异常条带泳动速度更快 ,说明序列比腺瘤中更短 ;MSI H与病人的年龄、性别、腺瘤发生部位和病理学亚型之间未见统计学差异 ,但MSI H组的平均年龄 (5 6 5 0± 1 1 38)低于MSI L组 (6 0 36±1 1 34) ,女性所占比率 (5 6 )明显高于男性 ,6例MSI H中无 1例组织学类型为管状腺瘤 ;各位点在MSI H组的MSI改变率明显高于MSI L组 ,在TGFβRⅡ (A) 1 0 、hMSH6、TCF4、BAT2 6等位点有明显差异 (P <0 0 5 ,其中BAT2 6的P <0 0 1 ) .可以推断 :在结直肠癌发生发展的早期即腺瘤阶段即可表现微卫星不稳定性 ;微卫星不稳定性可以随结直肠肿瘤的发展过程而发展 ,并且特定的微卫星位点的改变可能仅发生于肿瘤进程的特定阶段 ;在结直肠癌     

Genetic heterogeneity of variable number tandem repeats in thymidylate synthase gene in colorectal cancer patients

PURPOSE: To analyze the genetic variability in a variable number of tandem repeats (VNTR) in the thymidylate synthase (TS) enhancer promoter region and assess the influence of functional alterations in mismatch repair genes by analyzing constitutional and tumoral DNA from patients with colorectal adenocarcinoma with a high microsatellite instability (MSI-H) or microsatellite stability (MSS) status. PATIENTS AND METHODS: Patients who underwent surgery for colorectal adenocarcinoma were selected from the colorectal database of our institute and, on the basis of MSI status, assigned to a study group and a control group: group A, MSI-H; group B, MSS. Microsatellite status was investigated using the Bethesda recommended panel (BAT-26, BAT-25, D2S123, D5S346, D17S250). In MSI-H patients an additional analysis was made of the microsatellite loci D18S61 and D18S58, both mapping in the region containing the TS gene (18p11.2-11.32). Based on the number of altered microsatellites (> or = 2, 1, or 0), tumors were considered as having high (MSI-H) or low (MSI-L) instability or microsatellite stability (MSS), respectively. Genotyping for thymidylate synthase promoter polymorphism was carried out on constitutional and tumor DNA of each patient by PCR amplification of the polymorphic region. RESULTS: MSI-H was found in 55 patients (group A) and MSS in 50 patients (group B). In none of the MSI-H patients was microsatellite instability found in the additional D18S61 and D18S58 loci. In five group A and ten group B cases the analysis was not performed because constitutional DNA and/or tumoral DNA were not amplifiable. Homozygotes for the triple repeat variant (3R/3R) displayed only the large PCR product, homozygotes for the double repeat variant (2R/2R) displayed only the smaller PCR product, while heterozygotes (2R/3R) displayed both the larger and smaller PCR products. In 3/50 (6%) group A patients and 5/40 (12%) group B patients repeat variations were found in tumoral DNA. CONCLUSION: Our findings demonstrate that there is genetic homogeneity between constitutional and tumoral DNA but do not support the hypothesis that mismatch repair genes are involved in VNTR recombinant events in TS gene variability.     

A gene marker panel covering the Wnt and the Ras-Raf-MEK-MAPK signalling pathways allows to detect gene mutations in 80% of early (UICC I) colon cancer stages in humans

Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.     

Mutation analysis of hMSH2 and hMLH1 in colorectal cancer patients in India

The aim of this work was to study the mutation profile in hMSH2 and hMLH1 genes in hereditary nonpolyposis colorectal cancer (HNPCC) patients in India. On the basis of the Bethesda criteria, 31 colorectal cancer patients were studied first for microsatellite instability, using the five markers recommended by the Bethesda guidelines. Twelve of 31 tumor samples were found to be MSI-H, 9 of 31 were MSI-L, and the rest were MSS. The 12 patients with MSI-H were analyzed for mutations in hMSH2 and hMLH1 genes using PCR-denaturing high-performance liquid chromatography (dHPLC), followed by sequencing of samples showing abnormal peaks. Of the five mutations detected, three were found to be deleterious mutations (hMSH2-R680X, hMLH1-E671X, and a splice junction mutation IVS16-2A --> G); one had a mutation of probable significance (hMLH1-C680G) and one was of unknown significance (hMSH2-R171K). This study has also shown that most of the early-onset colon (4/7) and early-onset rectal (15/21) cancers are MSS or MSI-L. This is the first study to describe the mutation in hMSH2 and hMLH1 in Indian patients, a low incidence region for colorectal cancer. A two-stage procedure using MSI testing followed by PCR-dHPLC was found to be an efficient method in studying the mutation profile in high-risk patients.     

Assessment of microsatellite instability in head and neck cancer using consensus markers

Head and neck cancer is the sixth most common cancer in the world and one of the most lethal cancers. Microsatellite instability is an important characteristic of tumor cells and is observed both in presence and absence of mismatch repair gene mutations. The importance of microsatellite instability in head and neck cancer is not well established due to the lack of a consensus panel and selection of different markers, criteria and methodological variances. The main objective of this study was to investigate the performance of a consensus panel of microsatellite repeats by automated fragment analysis. Matched tumor and normal tissue samples from 99 patients were analyzed using five mononucleotide markers. Following PCR the amplified fragments were analyzed by capillary electrophoresis on an ABI 310 genetic analyzer. Microsatellite instability was observed in 26 patients. In 17 patients instability was detected at multiple loci. NR21 and BAT25 were the most frequently altered targets. These two mononucleotide markers could detect all samples displaying high-instability. In this study we describe a standardized fluorescent multiplex PCR combined with computerized analysis, which allows rapid and accurate analysis of a high number of samples and obviates the need to compare tumors with matching normal tissue.     

Mutations in exons 6 and 7 of TP53 gene correlate positively with serum tumor necrosis factor alpha independent of microsatellite instability in BAT26 gene in Egyptian patients with endometrial carcinoma

Abnormalities in the TP53 gene are the most frequent genetic alterations in human cancers. The role and mechanism of TP53 mutations have been well studied in many types of human cancer. Similarly, the presence of microsatellite instability (MSI) in the DNA mismatch repair system (hMSH2) may provide evidence of faulty DNA mismatch repair. One of the most important locations of MSI is the BAT26 gene. In addition, deranged serum cytokines, especially elevated levels of the tumor necrosis factor (TNF) alpha, have been found in many gynecological conditions. AIMS: The current study aimed at evaluating mutations in exons 6 and 7 of TP53 and the presence of microsatellite instability in BAT26 of the hMSH2 system in Egyptian patients with endometrial carcinoma. The study also evaluated whether there was a correlation between any of these genetic mutations/instability and the tissue expression of estrogen and progesterone receptors and the serum TNF-alpha level. PATIENTS AND METHODS: The current study included 2 groups: a control group comprising 20 healthy women aged 52.21 +/- 5.80 years attending the clinic for routine checkups and 40 patients with endometrial cancer aged 55.30 +/- 6.21 years. Mutations in TP53 and BAT26 were evaluated using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and automated sequencing while serum TNF-alpha was measured using an ELISA technique. Estrogen and progesterone receptor expression in biopsy tissue was evaluated using immunohistochemical staining. RESULTS: Seven of the 40 patients (17.5%) were positive for TP53 gene alterations in exon 6, while 9 patients (22.5%) were positive for TP53 alterations in exon 7. Cases positive for TP53 mutations had higher tumor stages. Ten patients (25%) showed MSI in BAT26. Nearly all patients with mutations in BAT26 had a strong family history for endometrial cancer (chi2=13.33, p<0.05). There was no positive correlation between the presence of MSI in the BAT26 gene and mutations in the TP53 gene or high serum TNF-alpha levels. Cases positive for TP53 mutations had a significantly higher level of TNF-alpha than cases negative for TP53 mutations (p<0.05). Cases showing mutations in exon 6 or 7 of TP53 showed a significantly higher intensity of immunohistochemical staining for estrogen and progesterone receptor expression in biopsy tissue than cases negative for mutations. (chi2=8.11, p<0.05). CONCLUSION: Our results suggest that the development of endometrial carcinoma is probably mediated through a multi-step carcinogenesis pathway and mutation of TP53 does not necessarily result from the presence of microsatellite instability in BAT26. The high serum TNF-alpha levels detected in our patients may represent an immunological antitumor response that was particularly evident in cases positive for TP53 mutations.     

Loss of heterozygosity on chromosome 5 in Iranian esophageal cancer patients

There is a high incidence of esophageal squamous cell carcinoma (ESCC) in Iran. Non-functionality of some tumor suppressor genes has been reported in esophageal cancer. Loss of heterozygosity on chromosome 5 has also been reported in esophageal carcinomas. We assessed loss of heterozygosity along a region of the long arm of chromosome 5 (5q), from 5q23.1 to 5q23.2, by PCR amplifying DNA fragments of tumor tissues from patients with ESCC and their corresponding normal samples. The PCR products were electrophoresed on 6% non-denaturing polyacrylamide gels, and band intensity was shown by silver staining. Of 40 patients with ESCC, 27, 25 and 36% of informative cases showed allelic losses at microsatellite markers D5S1384, D5S1478 and D5S1505, respectively. Two of the 40 patients studied had microsatellite instability at marker D5S1384. Based on the fact that loss of heterozygosity with more than 22% incidence for a specific marker cannot be regarded as a random event, we add support to previous reports concerning the presence of tumor suppressor genes in this chromosome region and that they affect esophageal cancer development. According to the data in NCBI UniSTS, the PCR product size of human DNA with primers of the D5S1505 marker ranges from 243 to 275 bp, containing about 20 repeats of the TAGA tetranucleotide, while the amplicon size of one allele of one of our cases was 207 bp, with about 10 repeats of the TAGA tetranucleotide, which would be the shortest sequence reported so far.     

应用荧光标记多重PCR对原发性胃癌进行微卫星不稳定性检测

为探讨 19号染色体微卫星不稳定性 (microsatelliteinstability ,MSI)在原发性胃癌发生中的作用 ,选择覆盖 19号染色体的 2 6个高分辨 (<5cM)微卫星标记 ,采用FAM、TET和HEX3种不同颜色的荧光染料标记微卫星引物 ,应用荧光标记多重PCR对 44例原发性胃癌及其正常对照组织进行扩增 ,产物在ABI3 77测序仪上经变性聚丙烯酰胺凝胶电泳 ,通过GeneScanTM及GenotyperTM 软件进行图像收集和MSI分析。同时采用PCR SSLP 银染技术检测了其中4个位点的MSI状态。 2 6个微卫星位点中 ,2 2个 (84 62 % )存在MSI,以D19S40 2MSI发生频率最高 (2 2 73 % ) ,平均MSI频率为 8 3 %。 44例胃癌中 ,3 4例 (77 2 7% )在一个或一个以上位点出现MSI。MSI作为胃粘膜癌变过程中一种可能的分子病理学机制 ,可作为胃癌分子检测的指标之一     

Granulocyte-macrophage-colony stimulating factor in patients with colorectal cancer

Granulocyte-macrophage-colony stimulating factor (GM-CSF) belongs to the group of glycoproteins called colony-stimulating factors (CSFs). It has been shown that the activity of CSFs is not limited to the hematopoietic cells but can also affect the proliferation of colon carcinoma cell lines. The purpose of this investigation was to compare the serum level of GM-CSF in colorectal cancer patients to a control group, to assess the level of GM-CSF in relation to the level of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), and to define the sensitivity, the specificity and the predictive values of GM-CSF in colorectal cancer. In this study, the serum level of tumour markers was measured in 30 patients with colorectal cancer and in 20 healthy subjects. GM-CSF was assayed using ELISA system, CEA and CA 19-9 were measured by MEIA. The serum levels of CEA, CA 19-9 and GM-CSF were higher in the patients with colorectal cancer than in the control group. The sensitivities of CEA (63%) and CA 19-9 (56%) were lower than the GM-CSF sensitivity (80%). The specificities of tumour markers were 70% (CEA, GM-CSF) and 75% for CA 19-9. The GM-CSF predictive v values were higher than the CEA and CA 19-9 values. These results suggest that GM-CSF may be useful as tumour marker in colorectal cancer, but further studies are needed.     

Hereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch-repair-gene mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer-susceptibility condition characterized by early onset colorectal cancer. Germ-line mutations in one of four DNA mismatch repair (MMR) genes, hMSH2, hMLH1, hPMS1, or hPMS2, are known to cause HNPCC. Although many mutations in these genes have been found in HNPCC kindreds complying with the so-called Amsterdam criteria, little is known about the involvement of these genes in families not satisfying these criteria but showing clear-cut familial clustering of colorectal cancer and other cancers. Here, we applied denaturing gradient-gel electrophoresis to screen for hMSH2 and hMLH1 mutations in two sets of HNPCC families, one set comprising families strictly complying with the Amsterdam criteria and another set in which at least one of the criteria was not satisfied. Interestingly, hMSH2 and hMLH1 mutations were found in 49% of the kindreds fully complying with the Amsterdam criteria, whereas a disease-causing mutation could be identified in only 8% of the families in which the criteria were not satisfied fully. In correspondence with these findings, 4 of 6 colorectal tumors from patients belonging to kindreds meeting the criteria showed microsatellite instability, whereas only 3 of 11 tumors from the other set of families demonstrated this instability. Although the number of tumors included in the study admittedly is small, the frequencies of mutations in the MMR genes show obvious differences between the two clinical sets of families. These results also emphasize the practical importance of the Amsterdam criteria, which provide a valid clinical subdivision between families, on the basis of their chance of carrying an hMSH2 or an hMLH1 mutation, and which bear important consequences for genetic testing and counseling and for the management of colorectal cancer families.     

Genomic instability in neoplasia

Recent studies have demonstrated novel alterations of microsatellite DNA in tumor tissue. The alterations, termed microsatellite instability or replication error phenotype, have now been observed in tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC), the Muir-Torre syndrome (MTS) and in an increasing number of sporadic tumors. These observations, along with the use of genetic linkage analysis, have led to the identification of at least four genetic susceptibility loci for HNPCC, hMSH2, hMLH1, hPMS1 and hPMS2, each of which are involved in DNA mismatch repair. For those tumors demonstrating microsatellite instability, several different phenotypes may exist, the significance of which is currently unknown. Defective DNA mismatch repair may have important implications for the mechanism of tumorigenesis and the clinical behavior of tumors.     

Genetic instability and CpG methylation in the 5'-flanking region of the PAI-1 gene in Chinese patients with gastric cancer

We explored a possible correlation of genetic instability and CpG methylation in the 5'-flanking region of the PAI-1 gene with clinicopathologic features of gastric cancer in Chinese patients and looked for molecular markers for diagnosing gastric tumor development. Microsatellite instability and loss of heterozygosity of the PAI-1 gene locus D7S515, D7S471 and pai-1 in 50 specimens of gastric cancer and relevant pericancerous tissues were detected by PCR-single strand conformation polymorphism (PCR-SSCP) with sliver staining. Methylation-specific PCR was used to detect CpG methylation in the 5'-flanking region of the PAI-1 gene. Microsatellite instability was significantly more common in the negative than in the positive serosa infiltration group of gastric cancer (42.86 vs 2.33%). The frequency of microsatellite instability was significantly lower in the cases with lymph node metastasis than in those without metastasis (18.18 vs 2.56%); however, it was significantly higher in the low differentiation group than that in the middle or high differentiation groups (21.05 vs 0.00%). CpG methylation in the 5'-flanking region of the PAI-1 gene did not differ significantly. Microsatellite instability and loss of heterozygosity of the PAI-1 gene apparently regulates the development of gastric cancer through different pathways. Microsatellite instability could be used as a molecular marker for the development of gastric cancer. CpG methylation in the 5'-flanking region of the PAI-1 gene appears not to be involved in the development of gastric cancer.     

High frequency of microsatellite instability in sporadic colorectal cancer patients in Iran

Microsatellite instability in sporadic colorectal cancer patients was assessed, and the clinicopathological associations were evaluated in northeastern Iran, which is a high-risk region for gastrointestinal malignancies. Microsatellite instability (MSI) status of tumoral tissue, compared to normal tissue, was assessed with a standard panel of MSI markers on paraffin-embedded surgically resected tissues from 67 consecutive sporadic colorectal cancer patients. Eleven of the patients were under 40 years old. Female patients were significantly younger than male patients (mean age 54.2 vs 62.1 years, P = 0.020). MSI analysis revealed 18 cases of MSI-H (26.9%), 11 MSI-L (16.4%) and 38 MSS (microsatellite stable tumors; 56.7%). While a greater proportion of patients consisted of males, 56.7 vs 43.3% females, MSI-H was more frequent in females (34.5 vs 21.5%). MSI was associated with proximal location of tumor (P = 0.003) and lower stages of tumor (P = 0.002), while MSS tumors were associated with node metastasis. MSI has a higher frequency in sporadic colorectal cancer patients, suggesting that molecular epidemiology of the genetic alterations involved in colorectal cancer carcinogenesis has a different pattern in the Iranian population, which deserves further epidemiological attention. The high frequency of MSI-H in this population suggests that we should look at microsatellite instability prior to chemotherapy to determine the most appropriate chemotherapeutic strategy in our population.     

Genetic Characteristics of Mitochondrial DNA Was Associated with Colorectal Carcinogenesis and Its Prognosis

Clinical value of mitochondrial DNA has been described in colorectal cancer (CRC). To clarify its role in colorectal carcinogenesis, mitochondrial microsatellite instability (mtMSI) and other markers were investigated in CRCs and their precancerous lesions, as a multitier genetic study. DNA was isolated from paired normal and tumoral tissues in 78 tubular adenomas (TAs), 34 serrated polyps (SPs), and 100 CRCs. mtMSI, nucleus microsatellite instability (nMSI), KRAS mutation, and BRAF mutation were investigated in these tumors and their statistical analysis was performed. mtMSI was found in 30% of CRCs and 21.4% of precancerous lesions. Mitochondrial copy number was higher in SPs than TAs and it was associated with mtMSI in low grade TAs. KRAS and BRAF mutations were mutually exclusive in TAs and SPs. CRCs with mtMSI showed shorter overall survival times than the patients without mtMSI. In CRCs without nMSI or BRAF mutation, mtMSI was a more accurate marker for predicting prognosis. The genetic change of mitochondrial DNA is an early and independent event in colorectal precancerous lesions and mtMSI and mitochondrial contents are associated with the tubular adenoma-carcinoma sequence, resulting in poor prognosis. This result suggested that the genetic change in mitochondrial DNA appears to be a possible prognosis marker in CRC.     

Molecular and Clinical Characteristics of MSH6 Variants: An Analysis of 25 Index Carriers of a Germline Variant

The MSH6 gene is one of the mismatch-repair genes involved in hereditary nonpolyposis colorectal cancer (HNPCC). Three hundred sixteen individuals who were known or suspected to have HNPCC were analyzed for MSH6 germline mutations. For 25 index patients and 8 relatives with MSH6 variants, molecular and clinical features are described. For analysis of microsatellite instability (MSI), the five consensus markers were used. Immunohistochemical analysis of the MLH1, MSH2, and MSH6 proteins was performed. Five truncating MSH6 mutations, of which one was detected seven times, were found in 12 index patients, and 10 MSH6 variants with unknown pathogenicity were found in 13 index patients. Fourteen (54%) of 26 colorectal cancers (CRCs) and endometrial cancers showed no, or only weak, MSI. Twelve of 18 tumors of truncating-mutation carriers and 3 of 17 tumors of missense-mutation carriers showed loss of MSH6 staining. Six of the families that we studied fulfilled the original Amsterdam criteria; most families with MSH6, however, were only suspected to have HNPCC. In families that did not fulfill the revised Amsterdam criteria, the prevalence of MSH6 variants is about the same as the prevalence of those in MLH1/MSH2. Endometrial cancer and/or atypical hyperplasia were diagnosed in 8 of 12 female carriers of MSH6 truncating mutations. Most CRCs were localized distally in the colon. Although, molecularly, missense variants are labeled as doubtfully pathogenic, clinical data disclose a great resemblance between missense-variant carriers and truncating-mutation carriers. We conclude that, in all patients suspected to have HNPCC, MSH6-mutation analysis should be considered. Neither MSI nor immunohistochemistry should be a definitive selection criterion for MSH6-mutation analysis.     

MSH2 c.1452-1455delAATG is a founder mutation and an important cause of hereditary nonpolyposis colorectal cancer in the southern Chinese population

Hereditary nonpolyposis colorectal cancer (HNPCC) accounts for approximately 2% of all colorectal cancer (CRC) cases and is the most common hereditary CRC syndrome. We have previously reported a high incidence of microsatellite instability (MSI) and germline mismatch repair (MMR) gene mutations in young Hong Kong Chinese with CRC. Ongoing studies at the Hereditary Gastrointestinal Cancer Registry in Hong Kong have revealed a unique germline MSH2 c.1452-1455delAATG mutation that has not been reported in other ethnic groups. Detailed analysis showed that this specific MSH2 mutation constituted 21% of all germline MMR gene mutations and 36% of all MSH2 germline mutations identified. We designed a specific PCR-based diagnostic test on paraffin-embedded tissues and identified this germline mutation in 2 (1.5%) of 138 consecutive patients with early-onset CRC (<46 years of age at diagnosis). Haplotype analysis was performed using 11 microsatellite markers located between D2S391 and D2S123. All 10 families had the same disease haplotype, suggesting a founder effect. These 10 families all originated from the Chinese province of Guangdong, which historically included Hong Kong. It is the most populous of the Chinese provinces, with a population of >93 million. Further analysis suggested that this founder mutation may date back to between 22 and 103 generations ago. The identification of this MSH2 founder mutation has important implications for the design of mutation-detection strategies for the southern Chinese population. Since there were major emigrations from Hong Kong and Guangdong province during the 19th and 20th centuries, this finding is also significant for Chinese communities worldwide.