Mechanism Of Gene Inactivation Research Articles

Genome-scale quantification and prediction of pathogenic stop codon readthrough by small molecules

Premature termination codons (PTCs) cause ~10–20% of inherited diseases and are a major mechanism of tumor suppressor gene inactivation in cancer. A general strategy to alleviate the effects of PTCs would be to promote translational readthrough. Nonsense suppression by small molecules has proven effective in diverse disease models, but translation into the clinic is hampered by ineffective readthrough of many PTCs. Here we directly tackle the challenge of defining drug efficacy by quantifying the readthrough of ~5,800 human pathogenic stop codons by eight drugs. We find that different drugs promote the readthrough of complementary subsets of PTCs defined by local sequence context. This allows us to build interpretable models that accurately predict drug-induced readthrough genome-wide, and we validate these models by quantifying endogenous stop codon readthrough. Accurate readthrough quantification and prediction will empower clinical trial design and the development of personalized nonsense suppression therapies.

CDH1 methylation analysis in invasive lobular breast carcinomas with and without gene mutation

The proposed role of CDH1 (E-cadherin gene) methylation as a mechanism of gene inactivation in invasive lobular carcinoma (ILC) remains inconclusive. For many years, CDH1 promoter hypermethylation has been regarded as a mechanism for gene inactivation in ILC. However, this assumption has primarily relied on non-quantitative assays, which have reported CDH1 methylation frequencies ranging from 26 to 93% at CpG sites within the island region. Few studies employing quantitative methods and covering CpG island shores, regions of relatively low CpG density situated proximal to conventional promoter CpGs, have been conducted, revealing lower percentages of methylation ranging from 0 to 51%. Therefore, using the quantitative pyrosequencing method, we examined CDH1 methylation in the island region and shores in E-cadherin deficient ILC cases (15 with CDH1 mutation and 22 non-mutated), 19 cases of invasive breast carcinomas non-special type (IBC-NSTs), and five cases of usual ductal hyperplasia (UDH). Our analysis revealed CDH1 methylation frequencies ranging from 3 to 64%, with no significant increase in methylation levels in any group of ILCs (median = 12%) compared to IBC-NST (median = 15%). In addition, considering the poorly studied association between the number of tumor-infiltrating lymphocytes (TILs) and CDH1 methylation in breast cancer, we undertook a thorough analysis within our dataset. Our findings revealed a positive correlation between CDH1 methylation and the presence of TILs (r = 0.5; p-value < 0.05), shedding light on an aspect of breast cancer biology warranting further investigation. These findings challenge CDH1 methylation as a CDH1 inactivation mechanism in ILC and highlight TILs as a potential confounding factor in gene methylation.

Minigene Splicing Assays Identify 20 Spliceogenic Variants of the Breast/Ovarian Cancer Susceptibility Gene RAD51C.

Simple SummaryLoss-of-function variants of the RAD51C gene are known to confer a risk of breast and ovarian cancers. In this study, we analyzed the impact of RAD51C variants on splicing, a highly regulated gene expression step by which introns are removed and exons are sequentially joined. Exon recognition is guided by specific sequences, the 3′ and 5′ splice sites, which define the exon boundaries. Variants of these sequences of susceptibility genes may lead to aberrant splicing and abnormal transcripts that may trigger a disease. Splicing can be tested using a biotechnological tool called minigenes, which mimic the human gene of interest. Thus, we checked 20 RAD51C splice-site variants using the minigene mgR51C_ex2-8. We found that they all disrupted the splicing mechanism, and 16 variants could be classified as likely pathogenic. Our findings are clinically actionable, and variant carriers may benefit from tailored prevention protocols and therapies.RAD51C loss-of-function variants are associated with an increased risk of breast and ovarian cancers. Likewise, splicing disruptions are a frequent mechanism of gene inactivation. Taking advantage of a previous splicing-reporter minigene with exons 2-8 (mgR51C_ex2-8), we proceeded to check its impact on the splicing of candidate ClinVar variants. A total of 141 RAD51C variants at the intron/exon boundaries were analyzed with MaxEntScan. Twenty variants were selected and genetically engineered into the wild-type minigene. All the variants disrupted splicing, and 18 induced major splicing anomalies without any trace or minimal amounts (<2.4%) of the minigene full-length (FL) transcript. Twenty-seven transcripts (including the wild-type and r.904A FL transcripts) were identified by fluorescent fragment electrophoresis; of these, 14 were predicted to truncate the RAD51C protein, 3 kept the reading frame, and 8 minor isoforms (1.1–4.7% of the overall expression) could not be characterized. Finally, we performed a tentative interpretation of the variants according to an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme, classifying 16 variants as likely pathogenic. Minigene assays have been proven as valuable tools for the initial characterization of potential spliceogenic variants. Hence, minigene mgR51C_ex2-8 provided useful splicing data for 40 RAD51C variants.

The Two-Hit Hypothesis Meets Epigenetics.

The landmark paper by Kane and colleagues was the first report of DNA methylation in the promoter of the human MLH1 gene in sporadic colon cancers with mismatch repair (MMR) deficiency. In both cell lines and primary tumors, promoter methylation was associated with loss of MLH1 protein expression and with a lack of mutations in the MLH1 coding region. Together with subsequent papers that showed that this methylation was directly responsible for loss of MLH1 expression and MMR deficiency, the observation expanded the two-hit hypothesis of tumor suppressor gene loss in cancer to include both genetic and epigenetic mechanisms of gene inactivation. More broadly, the paper contributed to normalization of the hypothesis of an epigenetic basis for cancer development. See related article by Kane and colleagues, Cancer Res 1997;57:808-11.

The Oligodendrocyte Transcription Factor 2 OLIG2 regulates transcriptional repression during myelinogenesis in rodents

OLIG2 is a transcription factor that activates the expression of myelin-associated genes in the oligodendrocyte-lineage cells. However, the mechanisms of myelin gene inactivation are unclear. Here, we uncover a non-canonical function of OLIG2 in transcriptional repression to modulate myelinogenesis by functionally interacting with tri-methyltransferase SETDB1. Immunoprecipitation and chromatin-immunoprecipitation assays show that OLIG2 recruits SETDB1 for H3K9me3 modification on the Sox11 gene, which leads to the inhibition of Sox11 expression during the differentiation of oligodendrocytes progenitor cells (OPCs) into immature oligodendrocytes (iOLs). Tissue-specific depletion of Setdb1 in mice results in the hypomyelination during development and remyelination defects in the injured rodents. Knockdown of Sox11 by siRNA in rat primary OPCs or depletion of Sox11 in the oligodendrocyte lineage in mice could rescue the hypomyelination phenotype caused by the loss of OLIG2. In summary, our work demonstrates that the OLIG2-SETDB1 complex can mediate transcriptional repression in OPCs, affecting myelination.

Methylation-mediated expression of SPARC is correlated with tumor progression and poor prognosis of breast cancer

Secreted protein acidic and rich in cysteine (SPARC) plays a crucial role in the formation and progression of tumors. DNA methylation has become increasingly recognized as a frequent event of epigenetic alterations and one of the primary mechanisms of gene inactivation. The study aims to investigate the status of DNA methylation and the biofunction of SPARC in breast cancer. The qRT-PCR, BGS, and MSP methods were respectively employed to measure the relative mRNA expression levels and methylation status of SPARC. Additionally, the effects of SPARC on cell proliferation, migration, and invasion were examined in SPARC overexpression and knockdown cells. Immunohistochemical staining and western blot assay were used to examine the protein expression of genes. The expression levels of SPARC were found to be higher in breast cancer tissues and most breast cancer cells. The expression levels of SPARC in MDA-MB-231 and MCF-7 cells were significantly reversed by 5-Aza-dC treatment. Furthermore, the high expression and promoter DNA hypomethylation of SPARC were detected in triple-negative breast cancer tissues, while no expression changes of SPARC were found in luminal A breast cancer tissues. Overexpression of SPARC dramatically promoted MCF-7 cells migration and invasion, while knockdown of SPARC inhibited MDA-MB-231 cells migration and invasion. SPARC was involved in the epithelial-mesenchymal transition (EMT) process of breast cancer cells. The expression levels of mesenchymal markers N-cadherin, Vimentin, and β-catenin were upregulated, while E-cadherin was downregulated in SPARC overexpressed breast cancer cells. Conversely, the expression levels of EMT-related genes demonstrated the opposite trend in SPARC knockdown cells. To conclude, high expression of SPARC regulated by promoter hypomethylation promotes breast cancer cells migration and invasion, thus SPARC may act as an oncogene and serve as a potential target for breast cancer therapy.

Whole genome and transcriptome analysis of genetic determinants associated with cyclophosphamide in vitro in pediatric acute leukemias

Introduction: The main objective was to implement the determinations of the ex vivo resistance to cyclophosphamide and to identify the genetic profile for pediatric patients with acute leukemias. Methods: In order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix). We performed also array-based comparative genomic hybridization using a SurePrint G3 Human CGH Microarray. Data was analyzed by bioinformatics tools. Verification of the relative expression level of 20 genes was carried out by qRT- PCR. Results: We observed a multitude of differentially expressed genes, e.g. ANXA1 (FC=3,04), BCL2A1 (FC=2,69), SERPINA1 (FC=2,12), DHRS7 (FC=2,13), PCDH9 (FC=- 4,58), TTC28 (FC=-2,25) and DUSP1 (FC=-2,91). The expression of genes that code for inflammation mediated by chemokine and cytokine signaling, Wnt, angiogenesis and integrin signaling and T cell activation pathways genes affect the sensitivity of leukemic blasts to cyclophosphamide. Transcriptome level changes are associated with chromosomal aberrations, especially located on chromosomes 8, 10, 14, 15, 16 and 22. Conclusion: Our work delineated genes with differentiated expression and recurrent copy number changes, and revealed novel amplified loci and frequent deletions in resistant to cyclophosphamide cells, which may guide future work aimed at identifying the relevant target genes. In particular, deletion seems to be a frequent mechanism of IFIT3 gene inactivation. ANXA1, SERPINA1, TCF7 and BCL2A1 may also be included among the candidate genes of resistance (Ontological analysis).

SERS-Based Assessment of MRD in Acute Promyelocytic Leukemia?

Acute promyelocytic leukemia (APL) is characterized by a unique chromosome translocation t(15;17)(q24;q21), which leads to the PML/RARA gene fusion formation. However, it is acknowledged that this rearrangement alone is not able to induce the whole leukemic phenotype. In addition, epigenetic processes, such as DNA methylation, may play a crucial role in leukemia pathogenesis. DNA methylation, catalyzed by DNA methyltransferases (DNMTs), involves the covalent transfer of a methyl group (-CH3) to the fifth carbon of the cytosine ring in the CpG dinucleotide and results in the formation of 5-methylcytosine (5-mC). The aberrant gene promoter methylation can be an alternative mechanism of tumor suppressor gene inactivation. Understanding cancer epigenetics and its pivotal role in oncogenesis, can offer us not only attractive targets for epigenetic treatment but can also provide powerful tools in monitoring the disease and estimating the prognosis. Several genes of interest, such as RARA, RARB, p15, p16, have been studied in APL and their methylation status was correlated with potential diagnostic and prognostic significance. In the present manuscript we comprehensively examine the current knowledge regarding DNA methylation in APL pathogenesis. We also discuss the perspectives of using the DNA methylation patterns as reliable biomarkers for measurable residual disease (MRD) monitoring and as a predictor of relapse. This work also highlights the possibility of detecting aberrant methylation profiles of circulating tumor DNA (ctDNA) through liquid biopsies, using the conventional methods, such as methylation-specific polymerase chain reaction (MS-PCR), sequencing methods, but also revolutionary methods, such as surface-enhanced Raman spectroscopy (SERS).

Aberrant DNA hypermethylation-silenced LINC00886 gene accelerates malignant progression of laryngeal carcinoma

BackgroundLong noncoding RNAs (lncRNAs) play crucial role in formation and progression of tumors. DNA methylation has become increasingly recognized as a frequent event of epigenetic alterations and one of the primary mechanisms of gene inactivation. The research aims to investigate the biofunction of a novel lncRNA in LSCC. MethodsqRT-PCR, BGS, and MSP methods were employed to measure the relative expression level and methylation status of LINC00886. Additionally, we examined the effects of LINC00886 on cells proliferation and invasion using LINC00886 over-expression. Nude mouse xenograft models were conducted to assess LINC00886 effects on LSCC growth in vivo. High-throughput sequencing technology and Western blot assay were carried out to have an in-depth study of the downstream target genes and signaling pathways in which LINC00886 may participate. ResultsThe remarkable downregulation of LINC00886 was observed in tumor tissues and laryngeal cancer cell lines. The significant decrease of LINC00886 was correlated with pathological grade in LSCC tissues. The expression level of LINC00886 in laryngeal cancer cell lines was significantly reversed by 5-Aza-dC. The occurrence of aberrant methylation events in the LINC00886 TSS was more responsible for the down-expression of LINC00886. Over-expression of LINC00886 dramatically mitigated cell proliferation, migration, and invasion in vitro as well as suppressed tumor growth in vivo. LINC00886 may be associated with VEGFA/PI3K/AKT signaling pathways and epithelial-mesenchymal transition (EMT) process. ConclusionsWe provide the first evidence of the involvement of LINC00886 in laryngeal carcinoma, which was downregulated due to methylation of the promoter region and served as tumor suppressor genes. LINC00886 is expected to become a novel biomarker in laryngeal carcinoma.

Clinical significance of aberrant DEUP1 promoter methylation in hepatocellular carcinoma.

Accumulating studies have shown that methylation of tumor suppressor genes plays an important role in tumorigenesis. Deuterosome assembly protein 1 (DEUP1) has been implicated as a suppressor gene in some tumors and promoter methylation led to silencing of its expression. However, the roles of DEUP1 promoter methylation and expression in hepatocellular carcinoma (HCC) are not clear. In the present study, the expression and methylation of the DEUP1 promoter in HCC was investigated and the correlations with HCC occurrence and development were explored. A total of 60 HCC tumor and adjacent non-tumor tissues were included in this study. Reverse transcription-polymerase chain reaction, bisulfite PCR sequencing, immunohistochemistry and western blotting were applied to detect the methylation status of the DEUP1 promoter and its expression, and to analyze their associations with clinicopathological data. The results showed that the mRNA and protein expression of DEUP1 in adjacent non-tumor tissues was significantly increased compared with in the HCC tissues. DEUP1 promoter methylation was detected in 46/60 (76.7%) tumor tissues and there was a negative correlation between promoter methylation and DEUP1 protein expression (P<0.05). Analysis of the clinicopathological data revealed that the mRNA and protein expression of DEUP1, and its promoter methylation status, was associated with tumor node metastasis stage and tumor differentiation. Taken together, the results of the present study suggested that methylation of the DEUP1 promoter maybe an important mechanism for gene inactivation and has a critical role in the occurrence and development of liver cancer.

Mis-splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays.

Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis-splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2 exons 2-9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD-based minigene with exons 2-9 was constructed and validated in MCF-7 cells, producing the expected transcript (1016-nt/V1-BRCA2_exons_2-9-V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE-variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF-7 cells. Thus, we identified nine spliceogenic ESE-rich intervals where ESE-variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty-three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double-effect. Notably, nine spliceogenic-ESE variants were located within ESE-containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G>A triggered the usage of a noncanonical GC-donor 4-nt upstream. Thirty-six variants that induced severe anomalies (>60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants. In conclusion, a large fraction of BRCA2 variants (∼64%) provoked splicing anomalies lending further support to the high prevalence of this disease-mechanism. The low accuracy of ESE-prediction algorithms may be circumvented by functional ESE-mapping that represents an optimal strategy to identify spliceogenic ESE-variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Epigenetic biomarkers for noninvasive detection of colorectal cancer.

45 Background: Aberrant DNA hypermethylation is known to be a major mechanism for inactivation of cancer-associated genes, including tumor suppressor genes, in colorectal cancer (CRC) and in other human cancers. Cancer-specific DNA methylation patterns of cell-free DNA (cfDNA) isolated from blood samples is a non-invasive method to obtain representative epigenetic information from solid tumors. In the present study, we identified and validated colorectal cancer-specific methylation markers for diagnosis of the disease with high sensitivity and specificity. We also compared the relative amount of DNA methylation at these target sites in relation to colorectal cancer stage. Methods: For marker validation, a total of 154 samples drawn from 68 subjects diagnosed with colorectal cancer (Stage I to IV), 42 healthy donors, 14 subjects with benign colorectal diseases, and 30 subjects diagnosed with other cancer types (breast, liver and lung cancer: 10 cases each) were obtained for a randomized, blinded study. Cell-free DNA was then extracted from the samples, bisulfite converted, and DNA methylation was quantified by using the IvyGene Platform. Results: By quantifying DNA methylation at the target sites, colorectal cancer samples were differentiated from samples drawn from healthy subjects or subjects with benign disease with an overall sensitivity of 93% (95% CI: 86-99) and specificity of 100% (95% CI: 85-100). All stages (I to IV) of colorectal cancer were identified with sensitivities ranging from 67% to 100%. None of the 30 samples drawn from subjects diagnosed with breast, liver or lung cancers were incorrectly identified as a colorectal cancer by the assay, for a calculated analytical specificity of 100%. Conclusions: These results demonstrate the high diagnostic potential of cfDNA methylation markers isolated from blood for the detection of colorectal cancer. Taken together, these findings establish the utility of methylation biomarkers for the detection of colorectal cancers as early as Stage I. In addition, a quantitative analysis of cfDNA provides an opportunity for non-invasive detection and monitoring of disease.

Epigenetic regulator UHRF1 inactivates REST and growth suppressor gene expression via DNA methylation to promote axon regeneration.

Injured peripheral sensory neurons switch to a regenerative state after axon injury, which requires transcriptional and epigenetic changes. However, the roles and mechanisms of gene inactivation after injury are poorly understood. Here, we show that DNA methylation, which generally leads to gene silencing, is required for robust axon regeneration after peripheral nerve lesion. Ubiquitin-like containing PHD ring finger 1 (UHRF1), a critical epigenetic regulator involved in DNA methylation, increases upon axon injury and is required for robust axon regeneration. The increased level of UHRF1 results from a decrease in miR-9. The level of another target of miR-9, the transcriptional regulator RE1 silencing transcription factor (REST), transiently increases after injury and is required for axon regeneration. Mechanistically, UHRF1 interacts with DNA methyltransferases (DNMTs) and H3K9me3 at the promoter region to repress the expression of the tumor suppressor gene phosphatase and tensin homolog (PTEN) and REST. Our study reveals an epigenetic mechanism that silences tumor suppressor genes and restricts REST expression in time after injury to promote axon regeneration.

Recurrent transcriptional loss of the PCDH17 tumor suppressor in laryngeal squamous cell carcinoma is partially mediated by aberrant promoter DNA methylation.

Protocadherins are cell-cell adhesion molecules encoded by a large family of genes. Recent reports demonstrate recurrent silencing of protocadherin genes in tumors and provide strong arguments for their tumor supresor functionality. Loss of protocadherins may contribute to cancer development not only by altering cell-cell adhesion, that is a hallmark of cancer, but also by enhancing proliferation and epithelial mesenchymal transition of cells via deregulation of the WNT signaling pathway. In this study we have further corroborated our previous findings on the involvement of PCDH17 in laryngeal squamous cell carcinoma (LSCC). We used bisulfite pyrosequencing to analyze a cohort of primary LSCC tumors for alterations in PCDH17 promoter DNA methylation as an alternative gene inactivation mechanism to the homozygous deletions reported earlier. Moreover, we analyzed primary LSCC samples by immunohistochemistry for PCDH17 protein loss. We identified recurrent elevation of PCDH17 promoter DNA methylation in 32/81 (40%) primary tumors (P < 0.001) and therein hypermethylation of 12 (15%) cases in contrast to no tumor controls (n = 24) that were all unmethylated. Importantly, DNA demethylation by decitabine has restored low level PCDH17 expression in LSCC cell lines. In conclusion, we provide a mechanistic explanation of recurrently observed PCDH17 silencing in LSCC by demonstrating the role of promoter methylation in this process. In light of these findings and recent reports showing that PCDH17 methylation is detectable in serum of cancer patients we suggest that testing PCDH17 DNA methylation might serve as a potential biomarker in LSCC.

Epigenetic modification of SALL1 as a novel biomarker for the prognosis of early stage head and neck cancer.

This study examined Sal-like protein (SALL)1 methylation profiles in head and neck squamous-cell carcinoma (HNSCC) patients at diagnosis and follow-up, and evaluated their prognostic significance and value as a biomarker. SALL1 expression was examined in a panel of cell lines by quantitative reverse transcription PCR (qRT-PCR). Promoter methylation was determined by quantitative methylation-specific polymerase chain reaction (qMSP) and was compared to the clinical characteristics of 205 samples. SALL1 promoter methylation was associated with transcriptional inhibition and was correlated with disease recurrence in 31.7% of cases, with an odds ratio of 1.694 (95% confidence interval: 1.093-2.626; P = 0.018) by multivariate Cox proportional hazard regression analysis. SALL1 promoter hypermethylation showed highly discriminatory receiver operator characteristic curve profiles that clearly distinguished HNSCC from adjacent normal mucosal tissue, and was correlated with reduced disease-free survival in early stage T1 and T2 patients (log-rank test, P < 0.001). SALL1 methylation was significantly correlated with the methylation status of both SALL3 and CDH1. This study suggests that CpG hypermethylation is a likely mechanism of SALL1 gene inactivation, supporting the hypothesis that SALL1 might play a role in HNSCC tumorigenesis and could serve as an important biomarker.

Neurofibromin C terminus-specific antibody (clone NFC) is a valuable tool for the identification of NF1-inactivated GISTs

An increasing body of evidence supports the involvement of NF1 mutations, constitutional or somatic, in the pathogenesis of gastrointestinal stromal tumors (GISTs). Due to the large size of the NF1 locus, the existence of multiple pseudogenes and the wide spectrum of mechanisms of gene inactivation, the analysis of NF1 gene status is still challenging for most laboratories. Here we sought to assess the efficacy of a recently developed neurofibromin-specific antibody (NFC) in detecting NF1-inactivated GISTs. NFC reactivity was analyzed in a series of 98 GISTs. Of these, 29 were ‘NF1-associated' (17 with ascertained NF1 mutations and 12 arising in the context of clinically diagnosed Neurofibromatosis type 1 syndrome and thus considered bona fine NF1 inactivated); 38 were ‘NF1-unrelated' (either wild-type or carrying non-pathogenic variants of NF1). Thirty-one additional GISTs with no available information on NF1 gene status or with NF1 gene variants of uncertain pathogenic significance were also included in the analysis. Cases were scored as NFC negative when, in the presence of NFC positive internal controls, no cytoplasmic staining was detected in the neoplastic cells. NFC immunoreactivity was lost in 24/29 (83%) NF1-associated GISTs as opposed to only 2/38 (5%) NF1-unrelated GISTs (P=3e−11). NFC staining loss significantly correlated (P=0.007) with the presence of biallelic NF1 inactivation, due essentially to large deletions or truncating mutations. NFC reactivity was instead retained in two cases in which the NF1 alteration was heterozygous and in one case where the pathogenic NF1 variant, although homo/hemizygous, was a missense mutation predicted not to affect neurofibromin half-life. Overall this study provides evidence that NFC is a valuable tool for identifying NF1-inactivated GISTs, thus serving as a surrogate for molecular analysis.

Transposase-driven rearrangements in human tumors.

A new study shows that aberrant DNA transposase activity promotes structural alterations that are clonally selected to drive tumor development. This discovery uncovers novel mechanisms of tumor-suppressor gene inactivation and highlights a new approach to cancer gene identification.

Relationship Between Human mutL Homolog 1 (hMLH1) Hypermethylation and Colorectal Cancer: A Meta-Analysis.

BackgroundHypermethylation of CpG islands in gene promoter regions is an important mechanism of gene inactivation in cancers. Promoter hypermethylation of human mutL homolog 1 (hMLH1) has been implicated in a subset of colorectal cancers that show microsatellite instability (MSI), while the connection of the epigenetic inactivation of hMLH1 in colorectal cancers remains unknown. The aim of this study was to evaluate the relationship between the promoter hypermethylation of hMLH1 and colorectal cancers by performing a meta-analysis.Material/MethodsEligible studies were identified through searching PubMed, Cochrane Library, Web of Science, and Google Scholar databases. R Software including meta packages was used to calculate the pooled and odds ratios (ORs) with corresponding confidence intervals (CIs). Funnel plots were also performed to evaluate publication bias.ResultsThis meta-analysis obtained 45 articles, including 4096 colorectal cancer patients, and identified a significant association between hMLH1 hypermethylation and colorectal cancer risk using the fixed-effects model (OR=8.3820; 95% CI, 6.9202~10.1527; z=21.7431; P<0.0001) and random effects model pooled (OR=10.0963; 95% CI, 6.1919~16.4626; z=9.2688; P<0.0001). The significant relationship was found in subgroup analyses.ConclusionsThe results of this meta-analysis show a significant association between hMLH1 hypermethylation and colorectal cancer risk.

Epigenetic silencing of SALL3 is an independent predictor of poor survival in head and neck cancer

BackgroundThis study examined Sal-like protein (SALL)3 methylation profiles of head and neck cancer (HNSCC) patients at diagnosis and follow-up and evaluated their prognostic significance and value as a biomarker. SALL3 expression was examined in a panel of cell lines by quantitative reverse transcription polymerase chain reaction (RT-PCR). The methylation status of the SALL3 promoter was examined by quantitative methylation-specific PCR.ResultsSALL3 promoter methylation was associated with transcriptional inhibition and was correlated with disease recurrence in 64.8% of cases, with an odds ratio of 1.914 (95% confidence interval: 1.157–3.164; P = 0.011) by multivariate Cox proportional hazard regression analysis. SALL3 promoter hypermethylation showed highly discriminatory receiver operator characteristic curve profiles that clearly distinguished HNSCC from adjacent normal mucosal tissue, and was correlated with reduced disease-free survival (DFS) (log-rank test, P = 0.01). Hypermethylation of tumor-related genes was higher among patients with SALL3 methylation than among those without methylation (P < 0.001). Furthermore, SALL3 hypermethylation was associated with expression of TET1, TET2, and DNMT3A genes.ConclusionsThis study suggests that CpG hypermethylation is a likely mechanism of SALL3 gene inactivation, supporting the hypothesis that the SALL3 gene may play a role in the tumorigenesis of HNSCC and may serve as an important biomarker.

F7 gene variants modulate protein levels in a large cohort of patients with factor VII deficiency. Results from a genotype-phenotype study.

Congenital factor VII (FVII) deficiency is a rare bleeding disorder caused by mutations in F7 gene with autosomal recessive inheritance. A clinical heterogeneity with poor correlation with FVII:C levels has been described. It was the objective of this study to identify genetic defects and to evaluate their relationships with phenotype in a large cohort of patients with FVII:C<50 %. One hundred twenty-three probands were genotyped for F7 mutations and three polymorphic variants and classified according to recently published clinical scores. Forty out of 123 patients (33 %) were symptomatic (43 bleedings). A severe bleeding tendency was observed only in patients with FVII:C<0.10 %. Epistaxis (11 %) and menorrhagia (32 % of females in fertile age) were the most frequent bleedings. Molecular analysis detected 48 mutations, 20 not reported in the F7 international databases. Most mutations (62 %) were missense, large deletions were 6.2 %. Compound heterozygotes/homozygotes for mutations presented lower FVII:C levels compared to the other classes (Chi2=43.709, p<0,001). The polymorphisms distribution was significantly different among the three F7 genotypic groups (Chi2=72.289, p<0,001). The presence of truncating mutations was associated with lowest FVII:C levels (Chi2=21.351, p=0.002). This study confirms the clinical and molecular variability of the disease and the type of symptoms. It shows a good correlation between the type of F7 mutation and/or polymorphisms and FVII:C levels, without a direct link between FVII:C and bleeding tendency. The results suggest that large deletions are underestimated and that they represent a common mechanism of F7 gene inactivation which should always be investigated in the diagnostic testing for FVII deficiency.