Molecular genetic analysis of 103 sporadic colorectal tumours in Czech patients

The Czech Republic has one of the highest incidences of colorectal cancer (CRC) in Europe. To evaluate whether sporadic CRCs in Czech patients have specific mutational profiles we analysed somatic genetic changes in known CRC genes (APC, KRAS, TP53, CTNNB1, MUTYH and BRAF, loss of heterozygosity (LOH) at the APC locus, microsatellite instability (MSI), and methylation of the MLH1 promoter) in 103 tumours from 102 individuals. The most frequently mutated gene was APC (68.9% of tumours), followed by KRAS (31.1%), TP53 (27.2%), BRAF (8.7%) and CTNNB1 (1.9%). Heterozygous germline MUTYH mutations in 2 patients were unlikely to contribute to the development of their CRCs. LOH at the APC locus was found in 34.3% of tumours, MSI in 24.3% and MLH1 methylation in 12.7%. Seven tumours (6.9%) were without any changes in the genes tested. The analysis yielded several findings possibly specific for the Czech cohort. Somatic APC mutations did not cluster in the mutation cluster region (MCR). Tumours with MSI but no MLH1 methylation showed earlier onset and more severe mutational profiles compared to MSI tumours with MLH1 methylation. TP53 mutations were predominantly located outside the hot spots, and transitions were underrepresented. Our analysis supports the observation that germline MUTYH mutations are rare in Czech individuals with sporadic CRCs. Our findings suggest the influence of specific ethnic genetic factors and/or lifestyle and dietary habits typical for the Czech population on the development of these cancers.     

Clinicopathologic features in colorectal cancer patients with microsatellite instability

The microsatellite instability (MSI) mutational pathway is critical to carcinogenesis in a small but significant proportion of colorectal cancers. While MSI is identified in most cancers in individuals with hereditary non-polyposis colorectal cancer, the majority of MSI tumors are found in individuals with sporadic disease. Colorectal cancers arising as a result of MSI have distinct clinicopathologic features distinguishing them from those with microsatellite stability. MSI colorectal cancers affect a larger percentage of women, are usually localized proximal to the splenic flexure, and have a higher incidence of synchronous and metachronous tumors. They are associated with a mucinous histology, tumor-infiltrating lymphocytes, a Crohn's-like inflammatory response, and a higher grade but lower stage. Overall survival is better in individuals with MSI. The benefit of chemotherapy in MSI colorectal cancers, with and without lymph node metastases, remains unclear.     

Microsatellite instability in pediatric high grade glioma is associated with genomic profile and differential target gene inactivation

High grade gliomas (HGG) are one of the leading causes of cancer-related deaths in children, and there is increasing evidence that pediatric HGG may harbor distinct molecular characteristics compared to adult tumors. We have sought to clarify the role of microsatellite instability (MSI) in pediatric versus adult HGG. MSI status was determined in 144 patients (71 pediatric and 73 adults) using a well established panel of five quasimonomorphic mononucleotide repeat markers. Expression of MLH1, MSH2, MSH6 and PMS2 was determined by immunohistochemistry, MLH1 was assessed for mutations by direct sequencing and promoter methylation using MS-PCR. DNA copy number profiles were derived using array CGH, and mutations in eighteen MSI target genes studied by multiplex PCR and genotyping. MSI was found in 14/71 (19.7%) pediatric cases, significantly more than observed in adults (5/73, 6.8%; p = 0.02, Chi-square test). MLH1 expression was downregulated in 10/13 cases, however no mutations or promoter methylation were found. MSH6 was absent in one pediatric MSI-High tumor, consistent with an inherited mismatch repair deficiency associated with germline MSH6 mutation. MSI was classed as Type A, and associated with a remarkably stable genomic profile. Of the eighteen classic MSI target genes, we identified mutations only in MSH6 and DNAPKcs and described a polymorphism in MRE11 without apparent functional consequences in DNA double strand break detection and repair. This study thus provides evidence for a potential novel molecular pathway in a proportion of gliomas associated with the presence of MSI.     

MLH1 Region Polymorphisms Show a Significant Association with CpG Island Shore Methylation in a Large Cohort of Healthy Individuals

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We previously demonstrated that SNPs (rs1800734, rs749072, and rs13098279) in the MLH1 gene region are associated with MLH1 promoter island methylation, loss of MLH1 protein expression, and microsatellite instability (MSI) in colorectal cancer (CRC) patients. Recent studies have identified less CpG-dense “shore” regions flanking many CpG islands. These shores often exhibit distinct methylation profiles between different tissues and matched normal versus tumor cells of patients. To date, most epigenetic studies have focused on somatic methylation events occurring within solid tumors; less is known of the contributions of peripheral blood cell (PBC) methylation to processes such as aging and tumorigenesis. To address whether MLH1 methylation in PBCs is correlated with tumorigenesis we utilized the Illumina 450 K microarrays to measure methylation in PBC DNA of 846 healthy controls and 252 CRC patients from Ontario, Canada. Analysis of a region of chromosome 3p21 spanning the MLH1 locus in healthy controls revealed that a CpG island shore 1 kb upstream of the MLH1 gene exhibits different methylation profiles when stratified by SNP genotypes (rs1800734, rs749072, and rs13098279). Individuals with wild-type genotypes incur significantly higher PBC shore methylation than heterozygous or homozygous variant carriers (p<1.1×10⁻⁶; ANOVA). This trend is also seen in CRC cases (p<0.096; ANOVA). Shore methylation also decreases significantly with increasing age in cases and controls. This is the first study of its kind to integrate PBC methylation at a CpG island shore with SNP genotype status in CRC cases and controls. These results indicate that CpG island shore methylation in PBCs may be influenced by genotype as well as the normal aging process.     

Clinical and Molecular Characterization of Brazilian Patients Suspected to Have Lynch Syndrome

Lynch syndrome (LS) accounts for 3–5% of all colorectal cancers (CRC) and is inherited in an autosomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous/metachronous tumors and extra-colonic tumors. Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five mismatch repair genes (MMR), mostly in MLH1 and MSH2, but also in MSH6, PMS1 and PMS2. To comprehensively characterize 116 Brazilian patients suspected for LS, we assessed the frequency of germline mutations in the three minor genes MSH6, PMS1 and PMS2 in 82 patients negative for point mutations in MLH1 and MSH2. We also assessed large genomic rearrangements by MLPA for detecting copy number variations (CNVs) in MLH1, MSH2 and MSH6 generating a broad characterization of MMR genes. The complete analysis of the five MMR genes revealed 45 carriers of pathogenic mutations, including 25 in MSH2, 15 in MLH1, four in MSH6 and one in PMS2. Eleven novel pathogenic mutations (6 in MSH2, 4 in MSH6 and one in PMS2), and 11 variants of unknown significance (VUS) were found. Mutations in the MLH1 and MSH2 genes represented 89% of all mutations (40/45), whereas the three MMR genes (MSH6, PMS1 and PMS2) accounted for 11% (5/45). We also investigated the MLH1 p.Leu676Pro VUS located in the PMS2 interaction domain and our results revealed that this variant displayed no defective function in terms of cellular location and heterodimer interaction. Additionally, we assessed the tumor phenotype of a subset of patients and also the frequency of CRC and extra-colonic tumors in 2,365 individuals of the 116 families, generating the first comprehensive portrait of the genetic and clinical aspects of patients suspected of LS in a Brazilian cohort.     

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.     

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.     

食管鳞癌MLH1基因改变及其与微卫星不稳定的关系

MLH1是位于3p21．3上的一个DNA错配修复基因,其异常与多种肿瘤相关。为探索食管鳞癌中MLH1基因改变及其与微卫星不稳定(MSI)的关联情况,采用微卫星分析和RT—PCR方法检测了14个微卫星标志在食管癌中的状况及MLH1转录水平的表达,发现35％的食管癌出现至少一个微卫星的不稳定,66．7％的肿瘤在MLH1基因内标志D3S1611位点表现为杂合性丢失,但是MLH1没有明显的mRNA表达下调。MSI与食管癌分期、分级、淋巴结转移、患者年龄和性别等参数及MLH1基因杂合性丢失(LOH)之间无统计学意义的相关性。这些结果表明：食管癌中MLH1存在较高频率的等位基因丢失,但其mRNA表达水平并无明显异常;所测微卫星标志的不稳定是食管癌的频发事件,与MLH1基因LOH不存在必然联系。     

Specific variants in the MLH1 gene region may drive DNA methylation, loss of protein expression, and MSI-H colorectal cancer

Background

We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability.

Methodology/Principal Findings

We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls) and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls) and from Seattle (591 cases, 629 controls). Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohisotchemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10⁻⁴ when the SNP was examined alone).

Conclusions/Significance

The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.     

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

Objective

Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.

Design

We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤50 years old (n = 188), a group of sporadic CRC >50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.

Results

Mean LINE-1 methylation levels (±SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test).

Conclusions

LINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC.     

An Integrative CGH,MSI and Candidate Genes Methylation Analysis of Colorectal Tumors

Background

Different DNA aberrations processes can cause colorectal cancer (CRC). Herein, we conducted a comprehensive molecular characterization of 27 CRCs from Iranian patients.

Materials and Methods

Array CGH was performed. The MSI phenotype and the methylation status of 15 genes was established using MSP. The CGH data was compared to two established lists of 41 and 68 cancer genes, respectively, and to CGH data from African Americans. A maximum parsimony cladogram based on global aberrations was established.

Results

The number of aberrations seem to depend on the MSI status. MSI-H tumors displayed the lowest number of aberrations. MSP revealed that most markers were methylated, except RNF182 gene. P16 and MLH1 genes were primarily methylated in MSI-H tumors. Seven markers with moderate to high frequency of methylation (SYNE1, MMP2, CD109, EVL, RET, LGR and PTPRD) had very low levels of chromosomal aberrations. All chromosomes were targeted by aberrations with deletions more frequent than amplifications. The most amplified markers were CD248, ERCC6, ERGIC3, GNAS, MMP2, NF1, P2RX7, SFRS6, SLC29A1 and TBX22. Most deletions were noted for ADAM29, CHL1, CSMD3, FBXW7, GALNS, MMP2, NF1, PRKD1, SMAD4 and TP53. Aberrations targeting chromosome X were primarily amplifications in male patients and deletions in female patients. A finding similar to what we reported for African American CRC patients.

Conclusion

This first comprehensive analysis of CRC Iranian tumors reveals a high MSI rate. The MSI tumors displayed the lowest level of chromosomal aberrations but high frequency of methylation. The MSI-L were predominantly targeted with chromosomal instability in a way similar to the MSS tumors. The global chromosomal aberration profiles showed many similarities with other populations but also differences that might allow a better understanding of CRC''s clinico-pathological specifics in this population.     

Clinical Significance of MLH1 Methylation and CpG Island Methylator Phenotype as Prognostic Markers in Patients with Gastric Cancer

Background

To improve the outcome of patients suffering from gastric cancer, a better understanding of underlying genetic and epigenetic events in this malignancy is required. Although CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) have been shown to play pivotal roles in gastric cancer pathogenesis, the clinical significance of these events on survival outcomes in patients with gastric cancer remains unknown.

Methods

This study included a patient cohort with pathologically confirmed gastric cancer who had surgical resections. A cohort of 68 gastric cancers was analyzed. CIMP and MSI statuses were determined by analyzing promoter CpG island methylation status of 28 genes/loci, and genomic instability at 10 microsatellite markers, respectively. A Cox’s proportional hazards model was performed for multivariate analysis including age, stage, tumor differentiation, KRAS mutation status, and combined CIMP/MLH1 methylation status in relation to overall survival (OS).

Results

By multivariate analysis, longer OS was significantly correlated with lower pathologic stage (P = 0.0088), better tumor differentiation (P = 0.0267) and CIMP-high and MLH1 3'' methylated status (P = 0.0312). Stratification of CIMP status with regards to MLH1 methylation status further enabled prediction of gastric cancer prognosis.

Conclusions

CIMP and/or MLH1 methylation status may have a potential to be prognostic biomarkers for patients with gastric cancer.     

Relationship of the methylenetetrahydrofolate reductase C677T polymorphism with microsatellite instability and promoter hypermethylation in sporadic colorectal cancer

The methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with the expression of a thermolabile enzyme with decreased activity that influences the pool of methyl-donor molecules. Several studies have reported an association between C677T polymorphism and susceptibility to colorectal cancer (CRC). Considering that methylation abnormalities appear to be important for the pathogenesis of CRC, we examined the correlation between the genotype of the MTHFR C677T polymorphism, hypermethylation of the promoter region of five relevant genes (DAPK, MGMT, hMLH1, p16(INK4a), and p14(ARF)), and microsatellite instability, in 106 patients with primary CRCs in Brazil. We did not find significant differences in the genotypic frequencies of the MTHFR C677T polymorphism when one or more loci were hypermethylated. However, we did find a significant excess of 677TT individuals among patients with CRC who had microsatellite instability. This strong association was independent of the methylation status of hMLH1 and of the biogeographical genomic ancestry of the patients. Although the mechanism responsible for the link between the C677T polymorphism and microsatellite instability was not apparent, this finding may provide a clue towards a better understanding of the pathogenesis of microsatellite instability in human colorectal cancer.     

Comprehensive Biostatistical Analysis of CpG Island Methylator Phenotype in Colorectal Cancer Using a Large Population-Based Sample

Background

The CpG island methylator phenotype (CIMP) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers) using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel.

Metholodology/Principal Findings

DNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16), CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF) and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight). In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the 5 markers), multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and β-catenin (CTNNB1) activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status.

Conclusions

Our study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also suggest that KRAS mutation is related with a random CpG island methylation pattern which may lead to CIMP-low tumors.     

Penetrance and expressivity of MSH6 germline mutations in seven kindreds not ascertained by family history

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by inherited mutations in DNA mismatch-repair genes, most commonly MLH1 or MSH2. The role MSH6 plays in inherited cancer susceptibility is less well defined. The aim of this study was to investigate the penetrance and expressivity of MSH6 mutations in kindreds ascertained through endometrial cancer probands unselected for family history. Detailed pedigrees were constructed for six MSH6 mutation carriers. All reported cancers and precancers were confirmed, and tissues were obtained when available. Tumors were analyzed for microsatellite instability (MSI) and for expression of MSH2, MLH1, and MSH6. MSH6 mutation status was determined for 59 family members. Of these 59 individuals, 19 (32%) had confirmed cancers and precancers. There was an excess of mutation carriers among the 19 affected family members (11 [58%] of 19) compared with those among the 40 unaffecteds (8 [20%] of 40, P=.0065, odds ratio = 5.5, 95% CI = 1.66-18.19). In four of the seven tumors analyzed from mutation carriers other than the probands, MSI and/or MMR protein expression was consistent with the involvement of MSH6. Overall estimated penetrance of the MHS6 mutations was 57.7%. Of the tumors in mutation carriers, 78% were part of the extended HNPCC spectrum. This study demonstrates that MSH6 germline mutations are, indeed, associated with increased cancer risk and that the penetrance of mutations may be higher than appreciated elsewhere. A combination of MSI and immunohistochemistry analyses may be helpful in screening for MSH6 mutation carriers.     

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.     

Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore

Background

Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region.

Methods

Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed.

Results

Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations.

Conclusions

Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.     

Reduced FHIT Expression Is Associated with Mismatch Repair Deficient and High CpG Island Methylator Phenotype Colorectal Cancer

Colorectal cancer (CRC) is a heterogeneous disease and a major contributor to world cancer mortality rates. Molecular subtypes of CRC have become standards for CRC classification and have established prognostic potential. Here, we attempt to corroborate and provide further insight pertinent to the fragile histidine triad (FHIT) gene in microsatellite instable (MSI), microsatellite stable (MSS), and CpG island methylator phenotype (CIMP) CRC subtypes. We employed array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA) techniques to survey genomic aberrations in FHIT gene and their effects on FHIT protein expression using immunohistochemistry (IHC) in a CRC cohort. We further studied FHIT protein expression by IHC in a larger CRC cohort defined for its mismatch repair (MMR) protein expression and genomic methylation profiles. Our results show FHIT genomic deletions centered in exons 4 and 5 in most of MSI-CRC samples. Moreover, we confirmed the significant association of FHIT protein expression diminution (p=0.035) with MSI-CRC. In the larger cohort, reduced FHIT protein expression was significantly associated with CIMP-high subtype of CRC (p=0.009) and loss of PMS2 protein expression (p=0.017). We conclude that FHIT expression may be a valuable marker for CRC subtyping, and its diagnostic, prognostic, and therapeutic potential should be perused.     

高频度微卫星不稳定性大肠癌"修饰型"微卫星变化与MLH1及KRAS基因突变密切相关

本研究通过方法学的改良和观察方式的创新试图阐明这种现象的原因.微卫星非传统的检测方法仅能实现微卫星定性检测,我所在的研究组开发了自动片段分析双荧光标识技术,提高了微卫星检测的感度和重复性,并实现了微卫星片段变化长度的定量.小于6碱基的微卫星变化被定义为修饰型微卫星不稳定,大于8碱基的变化被定义为跳跃型微卫星不稳定,它们的电泳谱截然不同.前者表现为在非肿瘤来源微卫星位点基础上的增加或减少,后者表现为距离非肿瘤微卫星片段远隔部位的新波形的出现.通过研究我们发现,在DNA错配修复缺陷细胞系及基因敲除大鼠自发肿瘤样本,仅有修饰型微卫星不稳定性检出;在人类DNA错配修复缺陷细胞系连续80次传代也没有检出跳跃型变化.跳跃型变化不能通过简单重复序列不稳定基础上的增加或减少的累加而获得.在76例散发大肠癌,我们检测了微卫星不稳定性,KRAS基因突变,并对高频度微卫星不稳定性病例的两个主要DNA错配修复基因MSH2和MLHl进行了全长测序.我们发现,在大肠癌,按频度的传统分类与按波形变化的分类有高度的一致性,高频度微卫星不稳定性病例均检测到跳跃型表现,低频度微卫星不稳定性都表现为修饰型变化.在12例高频度微卫星不稳定病例,有三例检出了跳跃型和修饰型同时存在微卫星不稳定的特殊表型,这3例均检出KRAS的突变,更有趣的是该3例病例也同时检出了DNA错配修复基因MLH1的变异.而在其他9例高频度微卫星不稳定病例,KRAS突变及MLH1、MSH2交变未检出.通过对突变谱的分析我们还发现,修饰型微卫星不稳定与KTAS基因12号密码子的转换型突变高度相关,而微卫星稳定的病例检出的KRAS基因12号密码子突变多为颠换型突变.修饰型微卫星不稳定表型检出的高频度转换突变可由DNA错配修复缺陷的分子背景解释.通过本研究,我们认为以波形为基础的微卫星不稳定新分型可能是解决目前微卫星研究领域矛盾的一个选项.一直公认为高频度微卫星不稳定性是"真正"的DNA错配修复缺陷表型,我们的研究提示实际上高频度微卫星的可能是多元的.修饰型微卫星不稳定与DNA错配修复缺陷直接关联,而跳跃型微卫星不稳定的原因尚未阐明.在高频度为微型不稳定中,携带修饰型变化的病例可以通过DNA错配修复系统缺陷来解释其病因.