Review of genetic and epigenetic alterations in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is associated with multiple risk factors and is believed to arise from pre-neoplastic lesions, usually in the background of cirrhosis. However, the genetic and epigenetic events of hepatocarcinogenesis are relatively poorly understood. HCC display gross genomic alterations, including chromosomal instability (CIN), CpG island methylation, DNA rearrangements associated with hepatitis B virus (HBV) DNA integration, DNA hypomethylation and, to a lesser degree, microsatellite instability. Various studies have reported CIN at chromosomal regions, 1p, 4q, 5q, 6q, 8p, 10q, 11p, 16p, 16q, 17p and 22q. Frequent promoter hypermethylation and subsequent loss of protein expression has also been demonstrated in HCC at tumor suppressor gene (TSG), p16, p14, p15, SOCS1, RIZ1, E-cadherin and 14-3-3 sigma. An interesting observation emerging from these studies is the presence of a methylator phenotype in hepatocarcinogenesis, although it does not seem advantageous to have high levels of microsatellite instability. Methylation also appears to be an early event, suggesting that this may precede cirrhosis. However, these genes have been studied in isolation and global studies of methylator phenotype are required to assess the significance of epigenetic silencing in hepatocarcinogenesis. Based on previous data there are obvious fundamental differences in the mechanisms of hepatic carcinogenesis, with at least two distinct mechanisms of malignant transformation in the liver, related to CIN and CpG island methylation. The reason for these differences and the relative importance of these mechanisms are not clear but likely relate to the etiopathogenesis of HCC. Defining these broad mechanisms is a necessary prelude to determine the timing of events in malignant transformation of the liver and to investigate the role of known risk factors for HCC.

Usefulness of MLPA in the detection of SHOX deletions

SHOX haploinsufficiency causes a wide spectrum of short stature phenotypes, such as Leri-Weill dyschondrosteosis (LWD) and disproportionate short stature (DSS). SHOX deletions are responsible for approximately two thirds of isolated haploinsufficiency; therefore, it is important to determine the most appropriate methodology for detection of gene deletion. In this study, three methodologies for the detection of SHOX deletions were compared: the fluorescence in situ hybridization (FISH), microsatellite analysis and multiplex ligation-dependent probe amplification (MLPA). Forty-four patients (8 LWD and 36 DSS) were analyzed. The cosmid LLNOYCO3`M`34F5 was used as a probe for the FISH analysis and microsatellite analysis were performed using three intragenic microsatellite markers. MLPA was performed using commercial kits. Twelve patients (8 LWD and 4 DSS) had deletions in SHOX area detected by MLPA and 2 patients generated discordant results with the other methodologies. In the first case, the deletion was not detected by FISH. In the second case, both FISH and microsatellite analyses were unable to identify the intragenic deletion. In conclusion, MLPA was more sensitive, less expensive and less laborious; therefore, it should be used as the initial molecular method for the detection of SHOX gene deletion. (C) 2010 Elsevier Masson SAS. All rights reserved.

Genetic heterogeneity in familial acute myelogenous leukemia: Evidence for a second locus at chromosome 16q21-23.2

The identification of genes responsible for the rare cases of familial leukemia may afford insight into the mechanism underlying the more common sporadic occurrences. Here we test a single family with 11 relevant meioses transmitting autosomal dominant acute myelogenous leukemia (AML) and myelodysplasia for linkage to three potential candidate loci. In a different family with inherited AML, linkage to chromosome 21q22.1-22.2 was recently reported; we exclude linkage to 21q22.1-22.2, demonstrating that familial AML is a heterogeneous disease. After reviewing familial leukemia and observing anticipation in the form of a declining age of onset with each generation, we had proposed 9p21-22 and 16q22 as additional candidate loci. Whereas linkage to 9p21-22 can be excluded, the finding of a maximum two-point LOD score of 2.82 with the microsatellite marker D16S522 at a recombination fraction theta = 0 provides evidence supporting linkage to 16q22. Haplotype analysis reveals a 23.5-cM (17.9-Mb) commonly inherited region among all affected family members extending from D16S451 to D1GS289, In order to extract maximum linkage information with missing individuals, incomplete informativeness with individual markers in this interval, and possible deviance from strict autosomal dominant inheritance, we performed nonparametric linkage analysis (NPL) and found a maximum NPL statistic corresponding to a P-value of .00098, close to the maximum conditional probability of linkage expected for a pedigree with this structure. Mutational analysis in this region specifically excludes expansion of the AT-rich minisatellite repeat FRA16B fragile site and the CAG trinucleotide repeat in the E2F-4 transcription factor. The ''repeat expansion detection'' method, capable of detecting dynamic mutation associated with anticipation, more generally excludes large CAG repeat expansion as a cause of leukemia in this family.

Lymph Node Involvement and Not the Histophatologic Subtype Is Correlated with Outcome After Resection of Adenocarcinoma of the Ampulla of Vater

Background Intestinal and pancreaticobiliary types of Vater`s ampulla adenocarcinoma have been considered as having different biologic behavior and prognosis. The aim of the present study was to determine the best immunohistochemical panel for tumor classification and to analyze the survival of patients having these histological types of adenocarcinoma. Method Ninety-seven resected ampullary adenocarcinomas were histologically classified, and the prognosis factors were analyzed. The expression of MUC1, MUC2, MUC5AC, MUC6, CK7, CK17, CK20, CD10, and CDX2 was evaluated by using immunohistochemistry. Results Forty-three Vater`s ampulla carcinomas were histologically classified as intestinal type, 47 as pancreaticobiliary, and seven as other types. The intestinal type had a significantly higher expression of MUC2 (74.4% vs. 23.4%), CK20 (76.7% vs. 29.8%), CDX2 (86% vs. 21.3%), and CD10 (81.4% vs. 51.1%), while MUC1 (53.5% vs. 82.9%) and CK7 (79.1% vs. 95.7%) were higher in pancreatobiliary adenocarcinomas. The most accurate markers for immunohistochemical classification were CDX2, MUC1, and MUC2. Survival was significantly affected by pancreaticobiliary type (p=0.021), but only lymph node metastasis, lymphatic invasion, and stage were independent risk factors for survival in a multivariate analysis. Conclusion The immunohistochemical expression of CDX2, MUC1, and MUC2 allows a reproducible classification of ampullary carcinomas. Although carcinomas of the intestinal type showed better survival in the univariate analysis, neither histological classification nor immunohistochemistry were independent predictors of poor prognosis.

Understanding the genetic diversity, spatial genetic structure and mating system at the hierarchical levels of fruits and individuals of a continuous Theobroma cacao population from the Brazilian Amazon

Understanding the mating patterns of populations of tree species is a key component of ex situ genetic conservation. In this study, we analysed the genetic diversity, spatial genetic structure (SGS) and mating system at the hierarchical levels of fruits and individuals as well as pollen dispersal patterns in a continuous population of Theobroma cacao in Para State, Brazil. A total of 156 individuals in a 0.56 ha plot were mapped and genotyped for nine microsatellite loci. For the mating system analyses, 50 seeds were collected from nine seed trees by sampling five fruits per tree (10 seeds per fruit). Among the 156 individuals, 127 had unique multilocus genotypes, and the remaining were clones. The population was spatially aggregated; it demonstrated a significant SGS up to 15m that could be attributed primarily to the presence of clones. However, the short seed dispersal distance also contributed to this pattern. Population matings occurred mainly via outcrossing, but selfing was observed in some seed trees, which indicated the presence of individual variation for self-incompatibility. The matings were also correlated, especially within ((r) over cap (p(m)) = 0.607) rather than among the fruits ((r) over cap (p(m)) = 0.099), which suggested that a small number of pollen donors fertilised each fruit. The paternity analysis suggested a high proportion of pollen migration (61.3%), although within the plot, most of the pollen dispersal encompassed short distances (28m). The determination of these novel parameters provides the fundamental information required to establish long-term ex situ conservation strategies for this important tropical species. Heredity (2011) 106, 973-985; doi:10.1038/hdy.2010.145; published online 8 December 2010

Adjuvant chemotherapy for stage II colod cancer

The use of adjuvant chemotherapy following resection for all patients with stage III colon cancer is now part of the standard of care around the world. Recent trials have led to changes in the standard regimens, which now include the use of oxaliplatin (Eloxatin) for most patients with stage III colon cancer. The addition of oxaliplatin has resulted in a 23% reduction in the risk of recurrence compared with fluorouracil/leucovorin alone, with a small but statistically significant survival benefit. Unfortunately, no adequately powered trial has determined whether adjuvant chemotherapy is beneficial for stage II patients, and its use is much more controversial. Most investigators agree that adjuvant chemotherapy has some activity against stage H disease. However, its impact on progression-free and overall survival remains highly controversial. Despite the lack of data, there is growing acceptance of an informal classification system, which stratifies stage II patients by risk on the basis of clinical data, as a guide for deciding whether to use adjuvant therapy. The only phase III clinical trial for stage H patients currently ongoing in the United States uses molecular classification as the basis for patient randomization.

Evaluation of computational methods for the reconstruction of HLA haplotypes

Human leukocyte antigen (HLA) haplotypes are frequently evaluated for population history inferences and association studies. However, the available typing techniques for the main HLA loci usually do not allow the determination of the allele phase and the constitution of a haplotype, which may be obtained by a very time-consuming and expensive family-based segregation study. Without the family-based study, computational inference by probabilistic models is necessary to obtain haplotypes. Several authors have used the expectation-maximization (EM) algorithm to determine HLA haplotypes, but high levels of erroneous inferences are expected because of the genetic distance among the main HLA loci and the presence of several recombination hotspots. In order to evaluate the efficiency of computational inference methods, 763 unrelated individuals stratified into three different datasets had their haplotypes manually defined in a family-based study of HLA-A, -B, -DRB1 and -DQB1 segregation, and these haplotypes were compared with the data obtained by the following three methods: the Expectation-Maximization (EM) and Excoffier-Laval-Balding (ELB) algorithms using the arlequin 3.11 software, and the PHASE method. When comparing the methods, we observed that all algorithms showed a poor performance for haplotype reconstruction with distant loci, estimating incorrect haplotypes for 38%-57% of the samples considering all algorithms and datasets. We suggest that computational haplotype inferences involving low-resolution HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 haplotypes should be considered with caution.

DNA mismatch repair gene methylation in gastric cancer in individuals from northern Brazil

Gastric cancer is one of the most common malignancies. DNA methylation is implicated in DNA mismatch repair genes deficiency. In the present study, we evaluated the methylation status of MLH1, MSH2, MSH6 and PMS2 in 20 diffuse- and 26 intestinal-type gastric cancer samples and 20 normal gastric mucosal of gastric cancer patients from Northern Brazil. We found that none of the nonneoplastic samples showed methylation of any gene promoter and 50% of gastric, cancer samples showed at least one methylated gene promoter. Methylation frequencies of MLH1, MSH2, MSH6 and PMS2 promoter were 21.74%, 17.39%, 0% and 28.26% respectively in gastric cancer samples. MLH1 and PMS2 methylation were associated with neoplastic samples compared to nonneoplastic ones. PMS2:? methylation was associated with diffuse- and intestinal-type cancer compared with normal controls. Intestinal-type cancer showed significant association with MLH1 methylation. Diffuse-type cancer was significantly associated with MSH2 methylation. Our findings show differential gene methylation in tumoral tissue, which allows us to conclude that methylation is associated with gastric carcinogenesis. Methylation of mismatch repair genes was associated with gastric carcinogenesis and may be a helpful tool for diagnosis, prognosis and therapies. However, MSH6 does not seem to be regulated by methylation in our samples.

Matrix metalloproteinase-9 genotypes and haplotypes are associated with multiple sclerosis and with the degree of disability of the disease

Multiple sclerosis (MS) is an autoimmune disease causing severe neurological disability. This study was carried out in order to determine whether the MMP-9 C(-1562)T and (CA)(13-25) polymorphisms are associated with MS. A total of 165 patients (92 whites/73 mulattos) and 191 controls (96 whites/95 mulattos) were enrolled in the study. While no difference in C(-1562)T polymorphism was observed between MS and healthy subjects, (CA)(n) genotypes and alleles were associated with MS. Moreover, the haplotypes are not associated with MS but seem to be relevant to the clinical status of MS. Thus the (CA)(n) polymorphism may contribute to MS susceptibility, but C(-1562)T and (CA)(n) haplotypes may modulate disease severity. (c) 2009 Elsevier B.V. All rights reserved.

Tumor necrosis factor region polymorphisms are associated with AIDS and with cytomegalovirus retinitis

Background: The tumor necrosis factor (TNF) gene is located within the highly polymorphic major histocompatibility complex region, exhibiting the -308 CA promoter region polymorphism and six microsatellites (TNFa-f) spanning the region nearby the TNF locus. Objective: In the present Study, we evaluated the frequency of -308 CA and TNFa-e polymorphisms and respective haplotypes (in chromosomal sequence: TNFd-TNFe-308GA-TNFc-TNFa-TNFb), in 222 patients with AIDS, 52 of whom exhibited cytomegalovirus retinitis, and in 202 healthy HIV-negative individuals. Method: TNF microsatellite and single nucleotide polymorphism typings were performed by PCR followed by polyacrylamide gel electrophoresis. Results: The TNF-308A allele and the 4-3-C-2-7-1 haplotype were associated with susceptibility to AIDS, whereas the TNFb4 allele and the 3-3-C-1-11-4 haplotype were associated with protection against AIDS development. The TNFc2 allele and the 4-1-G-2-2-1 haplotype, which contains the TNFc2 allele, were associated with cytomegalovirus retinitis. Conclusion: This study highlights that polymorphic sites spanning the region nearby the TNF locus are associated with AIDS per se and with cytomegalovirus retinitis in AIDS patients. (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins

Genetic analysis of forest species Eugenia uniflora L. through of newly developed SSR markers

Nine microsatellite loci for genetic analysis of three populations of the tropical tree Eugenia uniflora L. (pitanga or Brazilian cherry) from fragments of semideciduous forest were developed. We used the technique of building a (GA)(n) and (CA)(n) microsatellite-enriched library by capture with streptavidin-coated magnetic beads. We assessed the polymorphism of seven microsatellites in 84 mature trees found in three areas (Ribeir (a) over tildeo Preto, Tambau and S (a) over tildeo Jose do Rio Pardo), highly impacted by the agricultural practices, in a large region among Pardo river and Mogi-Guacu river basins, in state of S (a) over tildeo Paulo, Brazil. All loci were polymorphic, and the number of alleles was high, ranging from 6 to 24, with a mean of 14.4. All stands showed the same high level of genetic diversity (mean H(E) = 0.83) and a low genetic differentiation (mean F(ST) = 0.031), indicating that genetic diversity was higher within rather than among populations. Seven of the nine loci were highly variable, and sufficiently informative for E. uniflora. It was concluded that these new SSR markers can be efficiently used for gene flow studies.

A new candidate locus for bilateral perisylvian polymicrogyria mapped on chromosome Xq27

Polymicrogyria (PMG) is characterized by an excessive number of small and prominent brain gyri, separated by shallow sulci. Bilateral perisylvian polymicrogyria (BPP) is the most common form of PMG. Clinical signs include pseudobulbar paresis, mental retardation, and epilepsy. Familial forms of BPP have been described and a candidate locus was previously mapped to chromosome Xq28, distal do marker DXS8103. The objective of this study was to perform linkage analysis in one family segregating BPP. A total of 15 individuals, including 8 affected patients with BPP were evaluated. Family members were examined by a neurologist and subjected to magnetic resonance imaging scans. Individuals were genotyped for 18 microsatellite markers, flanking a 42.3 cM interval on ch Xq27-q28. Two-point and multipoint linkage analysis was performed using the LINKAGE package and haplotype reconstruction was performed by GENEHUNTER software. Our results showed a wide spectrum of clinical manifestations in affected individuals with BPP, ranging from normal to mild neurological abnormalities. Two-point linkage analysis yield a Zmax=2.06 at theta=0.00 for markers DXS1205 and DXS1227. Multipoint lod-scores indicate a candidate interval of 13 cM between markers DSXS1205 and DXS8043, on ch Xq27.2-Xq27.3. These results point to a new locus for BPP in a more centromeric location than previously reported. (C) 2008 Wiley-Liss, Inc.

HPP1: A transmembrane protein-encoding gene commonly methylated in colorectal polyps and cancers

Adenomas are the precursors of most colorectal cancers. Hyperplastic polyps have been linked to the subset of colorectal cancers showing DNA microsatellite instability, but little is known of their underlying genetic etiology. Using a strategy that isolates differentially methylated sequences from hyperplastic polyps and normal mucosa, we identified a 370-bp sequence containing the 5' untranslated region and the first exon of a gene that we have called HPP1. Rapid amplification of cDNA ends was used to isolate HPP1 from normal mucose. Using reverse transcription-PCR, HPP1 was expressed in 28 of 30 (93%) normal colonic samples but in only seven of 30 (23%) colorectal cancers (P < 0.001). The 5' region of HPP1 included a CpG island containing 49 CpG sites, of which 96% were found to be methylated by bisulfite sequencing of DNA from colonic tumor samples. By COBRA analysis, methylation was detected in six of nine (66%) adenomas, 17 of 27 (63%) hyperplastic polyps, and 46 of 55 (84%) colorectal cancers. There was an inverse relationship between methylation level and mRNA expression in cancers (r = -0.67; P < 0.001), and 5-aza-2-deoxycytidine treatment restored HPP1 expression in two colorectal cancer cell lines. In situ hybridization of HPP1 indicated that expression occurs in epithelial and stromal elements in normal mucosa but is silenced in both cell types in early colonic neoplasia. HPP1 is predicted to encode a transmembrane protein containing follistatin and epidermal growth factor-like domains. Silencing of HPP1 by methylation may increase the probability of neoplastic transformation.

Serrated route to colorectal cancer: back street or super highway?

Morphological and molecular studies are beginning to distinguish separate evolutionary pathways for colorectal cancer, The serrated pathway encompassing hyperplastic aberrant crypt foci, hyperplastic polyps. mixed polyps, and serrated adenoma is increasingly being linked with genetic alterations, including DNA methylation, DNA microsatellite instability, Ii-ras mutation, and loss of chromosome Ip, The importance of the serrated pathway has been underestimated in terms of its frequency and potential for rapid progression, Copyright (C) 2001 John Wiley & Sons, Ltd.

Hyperplastic polyposis - Association with colorectal cancer

Hyperplastic polyposis is a loosely defined syndrome initially thought not to confer a clinically important predisposition to colorectal cancer. The aim of the current study was to examine the clinical, histologic, and molecular features of a prospective series of cases meeting a strict definition of the condition. Twelve patients were identified, seven of whom had developed colorectal cancer. Most polyps were hyperplastic, but 11 patients also had polyps containing dysplasia as either serrated adenomas. mixed polyps, or traditional adenomas. The mean percentage of dysplastic polyps in patients with cancer was 35%, and in patients without cancer, 11%(p < 0.05). Microsatellite instability (MSI) was present in 3 of 47 hyperplastic polyps and two of right serrated adenomas. Kras was mutated in 8 of 47 hyperplastic polyps and two of eight serrated adenomas. No polyps showed loss of heterozygosity of chromosomes 5q, 1p, or 18q. Two of seven cancers showed a high level of MSI. It is concluded that hyperplastic polyposis is associated with a high risk of colorectal cancer. Hyperplastic polyps are the dominant type of polyp, but most cases have some dysplastic epithelium. A higher proportion of dysplastic polyps is associated with increased cancer risk. Clonal generic changes are observed in some hyperplastic polyps and serrated adenomas.