DNA mismatch repair and Lynch syndrome

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

GSTM1 and GSTT1 polymorphisms as modifiers of age at diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) in a homogeneous cohort of individuals carrying a single predisposing mutation

The variability in phenotype that occurs for so-called ‘single-gene disorders’ may be because of germline alterations in numerous primary and “modifier” genes. Within HNPCC families harbouring the same primary predisposing mutation, differences exist in the site of cancer, age of onset of disease symptoms and, consequently, survival until diagnosis of disease. The current study investigated a cohort of 129 individuals, from 13 different families, who harbour the identical nonsense mutation (C1528T) in the hMLH1 gene, predisposing them primarily to Lynch I syndrome. This cohort was screened for previously described polymorphisms in the glutathione-S-transferase genes, viz. GSTT1 and GSTM1. Male null carriers for both GSTT1 and GSTM1 were approximately three times more at risk of developing cancer at an earlier age when compared to non-null males. This work, particularly because of the relatively large “homogeneous” primary mutation cohort, provides evidence that genotypic changes distinct from the primary ‘HNPCC-causing’ mutation, influence the survival period until diagnosis of disease. It provides an impetus for expanding the study to include a wider range of candidate modifier genes. Such work may potentially lead to the development of individualised interval screening regimens for individuals with varying modifier genotypes—an attractive option in a resource-poor country.     

Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.     

Array comparative genomic hybridization based identification of key genetic alterations at 2p21-p16.3 (MSH2, MSH6, EPCAM), 3p23-p14.2 (MLH1), 7p22.1 (PMS2) and 1p34.1-p33 (MUTYH) regions in hereditary non polyposis colorectal cancer (Lynch syndrome) in the Kingdom of Saudi Arabia

Lynch syndrome is inherited in an autosomal dominant mode. Lynch syndrome is caused by impairment of one or more of the various genes (most frequently MLH1 and MSH2) involved in mismatch repair. In this study, whole genome comparative genomic hybridization array (array CGH) based genomic analysis was performed on twelve Saudi Lynch syndrome patients. A total of 124 chromosomal alterations (structural loss) were identified at mean log2 ratio cut off value of ±0.25. We also found structural loss in 2p21-p16.3, 3p23-p14.2, 7p22.1 and 1p34.1-p33 regions. These findings were subsequently validated by real time quantitative PCR showing downregulation of MSH2, MSH6, EPCAM, MLH1, PMS2 and MUTYH genes. These findings shall help in establishing database for alterations in mismatch repair genes underlying Lynch syndrome in Saudi population as well as to determine the incidence ratio of these disorders. Guided counselling will subsequently lead to the prevention and eradication of Lynch Syndrome in the local population.     

Association of single nucleotide polymorphisms of ERCC1 and XPF with colorectal cancer risk and interaction with tobacco use

We investigated the association between polymorphisms in excision repair cross-complementation group 1 (ERCC1) (rs3212986, rs2298881 and rs11615) and xeroderma pigmentosum-complementation group F (XPF) (rs2276466 and rs6498486) and risk of colorectal cancer. A 1:1 matched case–control study was conducted. Conditional regression analysis indicated that individuals carrying the ERCC1 rs3212986 TT genotype and T allele had a marginally increased risk of colorectal cancer when compared with subjects with the GG genotype. Similarly, subjects carrying the rs11615 TT genotype and T allele had a marginally increased risk of colorectal cancer when compared with those with the CC genotype. Stratified analysis revealed that individuals with rs3212986 TT who were current or former smokers had a significantly increased risk of colorectal cancer, and a significant interaction was found between this SNP and cigarette smoking. In conclusion, our study suggests that rs3212986 and rs11615 polymorphisms are associated with risk of colorectal cancer in a Chinese population, particularly in smokers. This finding could be useful in revealing the genetic characteristics of colorectal cancer, and suggests more effective strategies for prevention and treatment.     

Identification and molecular characterization of two novel mutations in COL1A2 in two Chinese families with osteogenesis imperfecta

Osteogenesis imperfecta (OI, also known as brittle bone disease) is caused mostly by mutations in two type Ⅰ collagen genes, COL1A1 and COL1A2 encoding the pro-α1 (Ⅰ) and pro-α2 (Ⅰ) chains of type Ⅰ collagen, respectively. Two Chinese families with autosomal dominant OI were identified and characterized. Linkage analysis revealed linkage of both families to COL1A2 on chromosome 7q21.3-q22.1. Mutational analysis was carried out using direct DNA sequence analysis. Two novel missense mutations, c.3350A＞G and c.3305G＞C, were identified in exon 49 of COL1A2 in the two families, respectively. The c.3305G＞C mutation resulted in substitution of a glycine residue (G) by an alanine residue (A) at codon 1102 (p.G1102A), which was found to be mutated into serine (S), argine (R), aspartic acid (D), or valine (V) in other families. The c.3350A＞G variant may be a de novo mutation resulting in p.Y1117C. Both mutations co-segregated with OI in respective families, and were not found in 100 normal controls. The G1102 and Y1117 residues were evolutionarily highly conserved from zebrafish to humans. Mutational analysis did not identify any mutation in the COX-2 gene (a modifier gene of OI). This study identifies two novel mutations p.G1102A and p.Y1117C that cause OI, significantly expands the spectrum of COL1A2 mutations causing OI, and has a significant implication in prenatal diagnosis of OI.     

Genetic analysis of hereditary multiple exostoses in Tunisian families: a novel frame-shift mutation in the <Emphasis Type="Italic">EXT1</Emphasis> gene

Hereditary multiple exostoses (HME) is an autosomal dominant orthopaedic disorder most frequently caused by mutations in the EXT1 gene. The aim of the present study is to determine the underlying molecular defect of HME in two multigenerational Tunisian families with 21 affected members and to examine the degree of intrafamilial variability. Linkage analysis was performed using three microsatellite markers encompassing the EXT1 locus and mutation screening was carried out by direct sequencing. In family 1, evidence for linkage to EXT1 was obtained on the basis of a maximum LOD score of 4.26 at θ = 0.00 with D8S1694 marker. Sequencing of the EXT1 revealed a heterozygous G > T transversion (c.1019G>T) in exon 2, leading to a missense mutation at the codon 340 (p.Arg340Leu). In family 2 we identified a novel heterozygous 1 bp deletion in the exon 1 (c.529_531delA) leading to a premature codon stop and truncated EXT1 protein expression (p.Lys177LysfsX15). This mutation was associated with the evidence of an intrafamilial clinical variability and considered to be a novel disease-causing mutation in the EXT1 gene. These findings provide additional support for the involvement of EXT1 gene in the HME disease.     

Rates and outcomes of testing for lynch syndrome in a national colorectal cancer screening programme

BackgroundLynch Syndrome (LS), the most common cause of hereditary colorectal cancer (CRC), is characterised by pathogenic variants in mismatch repair (MMR) genes. Universal testing of all CRCs for LS can increase detection. Rates and outcomes of testing in Ireland’s national CRC screening programme have not been examined previously.MethodsCRCs diagnosed at two screening sites between 2015 and 2020 were identified. Patient records were used to determine if CRCs had been tested for MMR deficiency and if detected, what downstream testing to rule out LS or genetic testing to confirm LS was undertaken.ResultsOver five years, 206 CRCs were diagnosed. Testing for LS was carried out for 100% of CRCs at site A and 69% of CRCs at site B. Of CRCs tested for LS, 14 (8%) were MMR deficient. After downstream testing for BRAF mutation or hypermethylation of MLH1, three CRCs were identified as potentially LS-related. Of these two individuals declined genetic testing and one was lost to follow-up.ConclusionsBy 2020 both sites had implemented universal testing of all CRCs for LS. A small number of individuals were identified as being eligible for genetic testing for LS, however those offered declined testing and one individual was lost to follow up. This highlights the importance of universal testing and the need for referral pathways to ensure all appropriate individuals are referred onwards to genetic services.     

Inactivation of the hMSH3 mismatch repair gene in bladder cancer

Deficiency in the DNA mismatch repair (MMR) is frequently involved in various cancers. The hMSH3 gene is one of the human MMR genes whose role in bladder cancer is not known. We hypothesized that down-regulation of the hMSH3 gene might be involved in bladder cancer. In this study we analyzed this gene with regard to frame-shift mutation, single nucleotide polymorphism (SNP), a 9bp repeat in exon 1, loss of heterozygosity (LOH), immunohistochemistry, and methylation status in 102 bladder cancer samples. Immunohistochemistry revealed that hMSH3 expression in bladder cancer was significant decreased compared to normal epithelium (p<0.0001). An inverse correlation with pathological grade was found. The frame-shift mutation in the (A) 8 tract was lacking in bladder cancer. There was no significantly difference between bladder cancer samples and healthy controls' with regard to SNP and the 9bp repeat. In bladder cancer, presence of the codon 222 polymorphism, LOH, and the 9bp repeats in exon 1 had a correlation with either pathological stage or pathological grade. Presence of the codon 1036 polymorphism had significant correlation with pathological stage and a trend to correlation with pathological grade. After 5-aza-dC treatment, MSH3 expression was significantly enhanced in TCC and UMUC bladder cancer cells when compared to untreated cells. This is the first report suggesting that genetic and epigenetic alterations in the human MSH3 gene might play a significant role in the progression of bladder tumors.     

Identification of three novel mutations in the COL2A1 gene in four unrelated Chinese families with spondyloepiphyseal dysplasia congenita

Introduction

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant skeletal dysplasia characterized by short stature, abnormal epiphyses, and flattened vertebral bodies. The condition occurs through a mutation in the COL2A1 gene that encodes the type II procollagen alpha1 chain (proalpha1 (II)).

Method and Results

We investigated nine affected individuals from four unrelated Chinese families with SEDC. We screened for COL2A1 gene mutations, and identified found four missense mutations (G447A, G456A, R789C and G1152D). The G447A, G456A and G1152D mutations are novel and the R789C mutation has been reported previously in several other studies with a strikingly similar phenotype.

Conclusions

Our study extends the mutation spectrum of SEDC and is helpful in early molecular diagnoses of SEDC.     

Analysis of mitochondrial genome revealed a rare 50 bp deletion and substitutions in a family with hypertension

We have sequenced the complete mtDNA of a family with hypertension (HT), type 2 diabetes (T2D) and coronary artery disease (CAD). Our analysis revealed two novel mutations (C3519T, G13204A); of which G13204A replaces valine to isoleucine. In silico analysis of a rare missense mutation (T8597C) showed a deleterious effect. We also observed a 50 bp deletion (m.298_347del50) in the hypervariable region II (HVSII) of all the individuals, who had a common maternal lineage. This (50 bp) deletion was not found in 17,785 individuals from different ethnic populations of India or in a variety of different disease phenotypes. We predict that the mtDNA mutations might be responsible for the HT. Analysis of POLG (polymerase gamma) gene revealed 14 variants which might be responsible for some of the mtDNA mutations seen in this family.     

HOTAIRM1 as a potential biomarker for diagnosis of colorectal cancer functions the role in the tumour suppressor

Recent studies have revealed many different long noncoding RNAs (lncRNA), however, the investigation for their function and clinical value as tumour biomarkers has scarcely begun. Here, we found that expression of HOTAIRM1 was reduced in colorectal cancer (CRC) tissues compared with matched normal tissues, and plasma HOTAIRM1 levels in CRC patients were less than in controls. The cut‐off point was chosen as 0.003 with a sensitivity of 64.00% and a specificity of 76.50% in the validation set. The performance of HOTAIRM1 was highly comparable to carcinoembryonic antigen (CEA), and better than CA19‐9 and CA125. The combined assay of HOTAIRM1 and CEA raised the sensitivity and specificity to 84.00%. HOTAIRM1 knockdown resulted in obvious changes in expression of the cell proliferation related to genes and promoted cell proliferation. HOTAIRM1 plays a role of tumour suppressor in CRC; Down‐regulation of HOTAIRM1 can serve as a biomarker for CRC, and combined HOTAIRM1 and CEA assay might provide a promising diagnosis for CRC.     

Sex-dependent correlation between survival and expression of genes related to the circadian oscillator in patients with colorectal cancer

ABSTRACT

Recent evidence supports the important role of the circadian system in cancer progression in humans. The aim of the present study is to evaluate clock (cry1, cry2 and per2) and clock-controlled (vascular endothelial growth factor-a, early growth response protein 1 and estrogen receptor β) gene expression in colorectal cancer and adjacent tissue and identify a possible link between survival of patients and expression of above mentioned genes. The study includes 64 patients of both sexes with previously diagnosed colorectal cancer. RNA was extracted from the tumor tissue and adjacent parts of the resected colon, and real-time PCR was used for detection of clock gene expression. Expression of cry2 and per2 was significantly downregulated in tumor tissue compared to adjacent tissues. After splitting of the cohort according to sex, we detected downregulated levels of cry2 and per2 in male patients, but not in females. Splitting of male and female sub-cohorts according to presence of metastases revealed significant donwregulation of cry2 expression in female patients without distant metastasis. Better survival rate was associated with low expression of cry2 in female patients. Moreover, we observed an increase in cry1 expression in female patients with distant metastases in tumor compared to adjacent tissue. Accordingly, women with high expression of cry1 in tumor tissue displayed worse survival, which was not observed in men. Taken together, expression of clock and clock-controlled genes in tumors of males and females clustered according to presence of distant metastases correlated with survival analysis. Studied clock-controlled genes also showed sex-dependent changes. Low expression of vegf-a in tumor correlated with better survival in men but not in women. High expression of estrogen receptor β mRNA was related to better survival in women but not in men. Low expression of vegf-a, egr1 and estrogen receptor β was associated with worse survival in women compared to men. Our data indicate sex-dependent associations between clock and clock-controlled gene expression in cancer tissue and patient’s survival prognosis.     

Rapid discrimination of colon cancer cells with single base mutation in KRAS gene segment using laser tweezers Raman spectroscopy

Laser tweezers Raman spectroscopy (LTRS) as a label‐free and noninvasive technology has been proven to be an ideal tool for analysis of single living cells, which provides important fingerprint information without interference from surrounding environments. For the first time, LTRS system was successfully used to examine the colon cancer cells with single base mutation in KRAS gene segment, including DKS‐8 (KRAS wild‐type [WT]) and DLD‐1 (KRAS mutant‐type [MT]), HKE‐3 (KRAS WT) and HCT‐116 (KRAS MT). Spectra changes of some vital biomolecules due to the gene mutation state were sensitively recorded by our home‐made LTRS system. As a result of the comparison between DKS‐8 and DLD‐1 cells, an index of 97.5% of correct classification was obtained by combining LTRS with principle component analysis coupled with linear discriminant analysis (PCA‐LDA) statistical analysis, while an index of 97.0% of correct classification was achieved between HKE‐3 and HCT‐116 cells. Moreover, between WT cells (DKS‐8 and HKE‐3) vs MT cells (DLD‐1 and HCT‐116), the index of correct classification was 81.2%, which was quite encouraging. Our preliminary results showed that the LTRS system coupled with PCA‐LDA analysis will have a great potential for further applications in the rapid and label‐free detection of circulating tumor cells in liquid biopsy.      

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics,and with biomarkers of one-carbon metabolism

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.     

A novel missense mutation of the ATP2C1 gene in a Chinese patient with Hailey-Hailey disease

Benign familial chronic pemphigus (Hailey–Hailey disease, HHD; MIM 169600) is a rare autosomal dominant hereditary disorder characterized by pruritic vesicles, painful erosions and scaly erythematous plaques at the sites of friction and flexures. Mutations in ATP2C1, which encoding the human secretory pathway Ca²⁺/Mn²⁺-ATPase protein 1 (hSPCA1), have been identified as the pathogenic gene of HHD. We found a novel, distinct, heterozygous mutation during study of a Chinese patient with HHD. We identified a C→T transition at nucleotide 1235 (p.Thr352IIe), in exon 13 of ATP2C1. This observation would be useful for genetic counseling and prenatal diagnosis for affected families and in expanding the repertoire of ATP2C1 mutations underlying HHD.     

Enhanced expression of a novel protein in human cancer cells: a potential aid to cancer diagnosis

Cap43 is a protein whose RNA is induced under conditions of severe hypoxia or prolonged elevations of intracellular calcium. Cap43 protein is expressed at low levels in normal tissues; however, in a variety of cancers, including lung, brain, melanoma, liver, prostate, breast, and renal cancers, Cap43 protein is overexpressed in cancer cells. The low level of expression of Cap43 in some normal tissues compared to their cancerous counterparts combined with the high stability of Cap43 protein and mRNA makes the Cap43 gene a new, important cancer marker. We hypothesize that the mechanism of Cap43 overexpression in cancer cells involves a state of hypoxia characteristic of cancer cells where the Cap43 protein becomes a signature for this hypoxic state. This revised version was published online in August 2006 with corrections to the Cover Date.