Methylation analysis of DCC gene in saliva samples is an efficient method for non-invasive detection of superficial hypopharyngeal cancer.

BACKGROUND: Advances in upper gastrointestinal endoscopic technology have enabled early detection and treatment of hypopharyngeal cancer. However, in-depth pharyngeal observations require sedation and are invasive. It is important to establish a minimally invasive and simple evaluation method to identify high-risk patients. METHODS: Eighty-seven patients with superficial hypopharyngeal cancer and 51 healthy controls were recruited. We assessed the methylation status of DCC, PTGDR1, EDNRB, and ECAD, in tissue and saliva samples and verified the diagnostic accuracy by methylation analyses of their promoter regions using quantitative methylation-specific PCR. RESULTS: Significant differences between cancer and their surrounding non-cancerous tissues were observed in the methylation values of DCC (p = 0.003), EDNRB (p = 0.001), and ECAD (p = 0.043). Using receiver operating characteristic analyses of the methylation values in saliva samples, DCC showed the highest area under the curve values for the detection of superficial hypopharyngeal cancer (0.917, 95% confidence interval = 0.864-0.970), compared with those for EDNRB (0.680) and ECAD (0.639). When the cutoff for the methylation values of DCC was set at ≥0.163, the sensitivity to detect hypopharyngeal cancer was 82.8% and the specificity was 90.2%. CONCLUSIONS: DCC methylation in saliva samples could be a non-invasive and efficient tool for early detection of hypopharyngeal cancer in high-risk patients.

Identification of specific codon 201 mutation of the DCC Gene in the colonoscopic specimen of colorectal cancer.

Objective: To identify the mutation in codon 201 of the deleted in colorectal cancer gene in colorectal cancer, and to correlate that mutation to the histopathological grading of colorectal cancer. METHODS: The cross-sectional study was conducted from February 2019 to February 2021 after approval from the ethics review board of the Dow University of Health Sciences, Karachi, and comprised biopsy-proven colorectal cancer patients regardless of age and gender. After histopathological reporting, formalin-fixed paraffin-embedded tissue blocks of colorectal cancer were used for deoxyribonucleic acid extraction, followed by polymerase chain reaction optimisation and deoxyribonucleic acid Sanger sequencing for mutational analysis. Data was analysed using SPSS 25. RESULTS: Of the 100 biopsy specimens assessed, 45(45%) were selected. Of them, 13(29%) samples failed to show any band on gel electrophoresis. The remaining 32(71%) samples were used for Sanger sequencing. Of these, 1(3%) sample did not sequence, while 31(97%) showed sequencing. All the sequenced samples identified a mutation in codon 201 of exon 3 in the deleted in colorectal cancer gene; 30(97%) showed homozygosity, and 1(3%) showed heterozygosity. No significant association of point mutation was noted with various demographic and clinicopathological parameters (p>0.05). Conclusion: The deleted in colorectal cancer gene's missense mutation in codon 201 was frequently observed in colorectal cancer patients.

Mirror Hand Movements Caused by a Deletion of the DCC Gene.

This case report describes the evaluation of 16-year-old twins with chromosomal microarray and mirror movements.

Corticolimbic DCC gene co-expression networks as predictors of impulsivity in children.

Inhibitory control deficits are prevalent in multiple neuropsychiatric conditions. The communication- as well as the connectivity- between corticolimbic regions of the brain are fundamental for eliciting inhibitory control behaviors, but early markers of vulnerability to this behavioral trait are yet to be discovered. The gradual maturation of the prefrontal cortex (PFC), in particular of the mesocortical dopamine innervation, mirrors the protracted development of inhibitory control; both are present early in life, but reach full maturation by early adulthood. Evidence suggests the involvement of the Netrin-1/DCC signaling pathway and its associated gene networks in corticolimbic development. Here we investigated whether an expression-based polygenic score (ePRS) based on corticolimbic-specific DCC gene co-expression networks associates with impulsivity-related phenotypes in community samples of children. We found that lower ePRS scores associate with higher measurements of impulsive choice in 6-year-old children tested in the Information Sampling Task and with impulsive action in 6- and 10-year-old children tested in the Stop Signal Task. We also found the ePRS to be a better overall predictor of impulsivity when compared to a conventional PRS score comparable in size to the ePRS (4515 SNPs in our discovery cohort) and derived from the latest GWAS for ADHD. We propose that the corticolimbic DCC-ePRS can serve as a novel type of marker for impulsivity-related phenotypes in children. By adopting a systems biology approach based on gene co-expression networks and genotype-gene expression (rather than genotype-disease) associations, these results further validate our methodology to construct polygenic scores linked to the overall biological function of tissue-specific gene networks.

Genetic Susceptibility of DCC Gene in Gallbladder Cancer in Kashmir and Meta-Analysis.

Deleted in colorectal carcinoma (DCC) A > G (rs714) is the most widely studied SNP of tumor suppressor DCC gene found to be associated with increased risk of various cancers. Therefore, the aim of present case control study was to investigate the role of DCC A > G (rs714) in gallbladder cancer (GBC) in Kashmir and to conduct a meta-analysis of DCC A > G (rs714) polymorphism to demonstrate the more accurate strength of these associations. Genotyping was done by PCR/RFLP and confirmed by sequencing in 100 GBC cases, and 150 controls. We also performed a comprehensive meta-analysis of 2223 subjects (1118 cases and 1105 controls) to evaluate the association between DCC A > G (rs714) polymorphisms and cancer. In present case control study DCC A > G (rs714) genotypes did not modulate the GBC cancer risk. Meta-analysis results showed that DCC A > G (rs714) is associated with increased overall cancer risk. DCC A > G (rs714) polymorphism conferred significant risk for cancer in dominant model but in recessive model P-value was at borderline. DCC A > G (rs714) genotype was associated with increased risk of cancer in Asians and Kashmiri population whereas no such association was observed in Europeans. The evidence in this meta-analysis supports a modest involvement of DCC A > G (rs714) tumoursupressor pathway genes in cancer susceptibility.

DCC mutation update: Congenital mirror movements, isolated agenesis of the corpus callosum, and developmental split brain syndrome.

The deleted in colorectal cancer (DCC) gene encodes the netrin-1 (NTN1) receptor DCC, a transmembrane protein required for the guidance of commissural axons. Germline DCC mutations disrupt the development of predominantly commissural tracts in the central nervous system (CNS) and cause a spectrum of neurological disorders. Monoallelic, missense, and predicted loss-of-function DCC mutations cause congenital mirror movements, isolated agenesis of the corpus callosum (ACC), or both. Biallelic, predicted loss-of-function DCC mutations cause developmental split brain syndrome (DSBS). Although the underlying molecular mechanisms leading to disease remain poorly understood, they are thought to stem from reduced or perturbed NTN1 signaling. Here, we review the 26 reported DCC mutations associated with abnormal CNS development in humans, including 14 missense and 12 predicted loss-of-function mutations, and discuss their associated clinical characteristics and diagnostic features. We provide an update on the observed genotype-phenotype relationships of congenital mirror movements, isolated ACC and DSBS, and correlate this to our current understanding of the biological function of DCC in the development of the CNS. All mutations and their associated phenotypes were deposited into a locus-specific LOVD (https://databases.lovd.nl/shared/genes/DCC).

Genetic unraveling of colorectal cancer.

Colorectal cancer is a common disease in both men and women (being the third most common cancer in men and the second most common among women) and thus represents an important and serious public health issue, especially in the western world. Although it is a well-established fact that cancers of the large intestine produce symptoms relatively earlier at a stage that can be easily cured by resection, a large number of people lose their lives to this deadly disease each year. Recent times have seen an important change in the incidence of colorectal cancer in different parts of the world. The etiology of colorectal cancer is multifactorial and is likely to involve the actions of genes at multiple levels along the multistage carcinogenesis process. Exhaustive efforts have been made out in the direction of unraveling the role of various environmental factors, gene mutations, and polymorphisms worldwide (as well as in Kashmir-"a valley of gastrointestinal cancers") that have got a role to play in the development of this disease so that antitumor drugs could be developed against this cancer, first, and, finally, the responsiveness or resistance to these agents could be understood for combating this global issue.

Evaluation of the methylation profile of exfoliated cell samples from patients with head and neck squamous cell carcinoma.

BACKGROUND: Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. The purposes of this study were to determine the methylation profile of exfoliated tumors cells collected from patients with head and neck squamous cell carcinoma (HNSCC) and to evaluate its prognostic significance. METHODS: The methylation profile and level of a 20-gene panel were evaluated by quantitative methylation-specific polymerase chain reaction (qMSP) in exfoliated tumor cell samples from 96 patients with HNSCC. RESULTS: CCNA1 (60.4%), DCC (54.2%), and TIMP3 (35.4%) were frequently methylated in these samples. Patients with exfoliated tumors cells positive for DCC methylation showed a trend toward a lower local recurrence-free survival. CONCLUSION: These findings indicate that a low invasive method could be used to access the methylation profile of exfoliated cells from patients with HNSCC. Moreover, our data provide evidence that hypermethylation of DCC could be useful as prognostic indicator for this malignancy.

ATP-dependent roX RNA remodeling by the helicase maleless enables specific association of MSL proteins.

Dosage compensation in Drosophila involves a global activation of genes on the male X chromosome. The activating complex (MSL-DCC) consists of male-specific-lethal (MSL) proteins and two long, noncoding roX RNAs. The roX RNAs are essential for X-chromosomal targeting, but their contributions to MSL-DCC structure and function are enigmatic. Conceivably, the RNA helicase MLE, itself an MSL subunit, is actively involved in incorporating roX into functional DCC. We determined the secondary structure of roX2 and mapped specific interaction sites for MLE in vitro. Upon addition of ATP, MLE disrupted a functionally important stem loop in roX2. This RNA remodeling enhanced specific ATP-dependent association of MSL2, the core subunit of the MSL-DCC, providing a link between roX and MSL subunits. Probing the conformation of roX in vivo revealed a remodeled stem loop in chromatin-bound roX2. The active remodeling of a stable secondary structure by MLE may constitute a rate-limiting step for MSL-DCC assembly.

Deleted in Colorectal Cancer (DCC) gene polymorphism is associated with H. pylori infection among susceptible Malays from the north-eastern region of Peninsular Malaysia.

BACKGROUND/AIMS: Using genome-wide case-control association approach, the current study aimed to determine whether genetic polymorphism(s) is/are associated with H. pylori infection among ethnic Malays from the north-eastern region of Peninsular Malaysia, a region with an exceptionally low prevalence for H. pylori infection and gastric cancer. METHODOLOGY: Twenty-three Malay subjects positive for H. pylori confirmed with urease test and histology were enrolled as "cases" and 37 subjects negative for H. pylori were "controls". Both groups were matched for age and environmental risks. Extracted DNA samples (QIAGEN, Germany) from the venous blood of study subjects were genotyped using the Human Mapping 50k xbal array (Affymetrix, USA). High throughput downstream analyses were then used to determine the significant SNP(s) associated with H. pylori infection. RESULTS: Out of 20,361 SNPs filtered using the genotype association test, the top 1% (203) significant SNPs were selected for functional enrichment analysis. Of the 15 "enriched" SNPs, the rs10502974 which was located within the intronic region of Deleted in Colorectal Cancer (DCC) gene was the SNP most significantly associated with H. pylori infection (p=0.00549). CONCLUSIONS: Ethnic Malays is genetically susceptible to H. pylori infection and is possibly mediated through a genetic variation in the DCC gene.

Deleted in Colorectal Cancer (DCC) pathfinding: axon guidance gene finally turned tumor suppressor.

Loss of heterozygosity (LOH) at human chromosome 18q, which includes the gene Deleted in Colorectal Cancer (DCC), has been linked to colorectal and many other human cancers. DCC encodes the receptor for the axon guidance molecule Netrin (Net) and functions during neural development in a variety of organisms. However, since its discovery in the 1990s, the status of DCC as a tumor suppressor has been debated, primarily due to a lack of support for this hypothesis in animal models. A recent study from our laboratory capitalized on the genetic tractability of Drosophila melanogaster to demonstrate that this gene functions as an invasive tumor suppressor, thereby providing the first direct link between DCC loss and metastatic phenotypes in an animal model for cancer. Two subsequent studies from other laboratories have demonstrated that DCC suppresses tumor progression and metastasis in murine colorectal and mammary tumor models. Combined, these findings have prompted the rebirth of DCC as a tumor suppressor and highlighted the need for continued analysis of DCC function in animal models for human cancer.

Comparison of promoter hypermethylation pattern in salivary rinses collected with and without an exfoliating brush from patients with HNSCC.

BACKGROUND: Salivary rinses have been recently proposed as a valuable resource for the development of epigenetic biomarkers for detection and monitoring of head and neck squamous cell carcinoma (HNSCC). Both salivary rinses collected with and without an exfoliating brush from patients with HNSCC are used in detection of promoter hypermethylation, yet their correlation of promoter hypermethylation has not been evaluated. This study was to evaluate the concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush from patients with HNSCC. METHODOLGY: 57 paired salivary rinses collected with or without an exfoliating brush from identical HNSCC patients were evaluated for promoter hypermethylation status using Quantitative Methylation-Specific PCR. Target tumor suppressor gene promoter regions were selected based on our previous studies describing a panel for HNSCC screening and surveillance, including P16, CCNA1, DCC, TIMP3, MGMT, DAPK and MINT31. PRINCIPAL FINDINGS: In salivary rinses collected with and without brush, frequent methylation was detected in P16 (8.8% vs. 5.2%), CCNA1 (26.3% vs. 22.8%), DCC (33.3% vs. 29.8%), TIMP3 (31.6% vs. 36.8%), MGMT (29.8% vs. 38.6%), DAPK (14.0% vs. 19.2%), and MINT31 (10.5% vs. 8.8%). Spearman's rank correlation coefficient showed a positive correlation between salivary rinses collected with and without brush for P16 (ρ = 0.79), CCNA1 (ρ = 0.61), DCC (ρ = 0.58), TIMP3 (ρ = 0.10), MGMT (ρ = 0.70), DAPK (ρ = 0.51) and MINT31 (ρ = 0.72) (P<0.01). The percent agreement of promoter methylation between salivary rinses with brush and without brush were 96.5% for P16, 82.5% for CCNA1, 78.9% for DCC, 59.7% for TIMP3, 84.2% for MGMT, 84.2% for DAPK, and 94.7% for MINT31. CONCLUSIONS: Our study demonstrated strong correlations of gene promoter hypermethylation between salivary rinses collected with and without an exfoliating brush. Salivary rinse collection without using an exfoliating brush may offer a cost effective, rapid, non-invasive, and reliable means for development of epigenetic salivary rinse biomarkers.

Detection of TIMP3 promoter hypermethylation in salivary rinse as an independent predictor of local recurrence-free survival in head and neck cancer.

PURPOSE: To validate a panel of methylation-based salivary rinse biomarkers (P16, CCNA1, DCC, TIMP3, MGMT, DAPK, and MINT31) previously shown to be independently associated with poor overall survival and local recurrence in a larger, separate cohort of patients with head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: One hundred ninety-seven patients were included. All pretreatment saliva DNA samples were evaluated for the methylation status of the gene promoters by quantitative methylation-specific PCR. The main outcome measures were overall survival, local recurrence-free survival, and disease-free survival. RESULTS: In univariate analyses, the detection of hypermethylation of CCNA1, MGMT, and MINT31 was significantly associated with poor overall survival; the detection of hypermethylation of TIMP3 was significantly associated with local recurrence-free survival; and the detection of hypermethylation of MINT31 was significantly associated with poor disease-free survival. In multivariate analyses, detection of hypermethylation at any single marker was not predictive of overall survival in patients with HNSCC; detection of hypermethylation of TIMP3 in salivary rinse had an independent, significant association with local recurrence-free survival (HR = 2.51; 95% CI: 1.10-5.68); and none of the studied markers was significantly associated with disease-free survival. CONCLUSION: The detection of promoter hypermethylation of the seven genes in salivary rinse as an independent prognostic indicator of overall survival in patients with HNSCC was not validated. Detection of promoter hypermethylation of TIMP3 in pretreatment salivary rinse is independently associated with local recurrence-free survival in patients with HNSCC and may be a valuable salivary rinse biomarker for HNSCC recurrence.

Ki-ras gene mutations are invariably present in low-grade mucinous tumors of the vermiform appendix.

OBJECTIVE: Low-grade mucinous tumors of the appendix appear to have a simple histological structure. Paradoxically, reports have suggested a greater frequency of Ki-ras gene mutation in these lesions than in more complex lesions such as benign colonic adenomas and carcinomas. We assessed several molecular genetic changes, including Ki-ras gene mutations, in a large series of low-grade mucinous tumors of the appendix. MATERIAL AND METHODS: We retrospectively ascertained low-grade mucinous tumors of the appendix from computerized pathology records. Extracted DNA was analyzed for APC and DCC gene loss of heterozygosity, microsatellite instability and for the presence of Ki-ras gene mutation using standard molecular techniques. Controls consisted of normal appendices, other appendiceal neoplasms, and ovarian mucinous cystadenomas. RESULTS: A total of 31 low-grade appendiceal mucinous tumors were identified. All were microsatellite stable and none demonstrated loss of heterozygosity for the APC or DCC genes. By contrast, all 31 lesions contained a Ki-ras gene mutation. CONCLUSIONS: The presence of a Ki-ras gene mutation in all lesions, with no other molecular changes identified, strongly suggests a possible etiological role of the Ki-ras mutation in the development of this particular lesion of the appendix. Based on other work regarding intestinal bacteria, we hypothesize a relationship between chronic inflammation of the appendix from bacterial overgrowth and Ki-ras gene mutation.

DCC is specifically required for the survival of retinal ganglion and displaced amacrine cells in the developing mouse retina.

Netrin-1 and DCC are well known for their roles in neurite growth, axonal guidance, and neuronal migration. Recently, a number of studies showed that DCC is involved in the induction of apoptosis, and this proapoptotic activity can be blocked in the presence of Netrin-1. However, here, we found that DCC is required for the survival of two types of neurons selectively in the developing mouse retina where DCC is abundantly expressed. Our results showed that the DCC(-/-) retina displayed a reduced ganglion cell layer with relatively normal neuroblastic layer. Immunostaining assays revealed that in DCC(-/-) mice, initial neurogenesis within retina was unchanged while the numbers of differentiated retinal ganglion cells and displaced amacrine cells in ganglion cell layer were greatly reduced due to increased apoptosis. By contrast, other neuronal types including horizontal cells, bipolar cells, amacrine cells, photoreceptors, and Müller cells appeared normal in DCC mutant retinas. Moreover, DCC(kanga) mice that lack the intracellular P3 domain of DCC receptor displayed the same defects as DCC(-/-) mice. Thus, our findings suggest that DCC is a key regulator for the survival of specific types of neurons during retinal development and that DCC-P3 domain is essential for this developing event.

Distinguishing between cancer driver and passenger gene alteration candidates via cross-species comparison: a pilot study.

BACKGROUND: We are developing a cross-species comparison strategy to distinguish between cancer driver- and passenger gene alteration candidates, by utilizing the difference in genomic location of orthologous genes between the human and other mammals. As an initial test of this strategy, we conducted a pilot study with human colorectal cancer (CRC) and its mouse model C57BL/6J ApcMin/+, focusing on human 5q22.2 and 18q21.1-q21.2. METHODS: We first performed bioinformatics analysis on the evolution of 5q22.2 and 18q21.1-q21.2 regions. Then, we performed exon-targeted sequencing, real time quantitative polymerase chain reaction (qPCR), and real time quantitative reverse transcriptase PCR (qRT-PCR) analyses on a number of genes of both regions with both human and mouse colon tumors. RESULTS: These two regions (5q22.2 and 18q21.1-q21.2) are frequently deleted in human CRCs and encode genuine colorectal tumor suppressors APC and SMAD4. They also encode genes such as MCC (mutated in colorectal cancer) with their role in CRC etiology unknown. We have discovered that both regions are evolutionarily unstable, resulting in genes that are clustered in each human region being found scattered at several distinct loci in the genome of many other species. For instance, APC and MCC are within 200 kb apart in human 5q22.2 but are 10 Mb apart in the mouse genome. Importantly, our analyses revealed that, while known CRC driver genes APC and SMAD4 were disrupted in both human colorectal tumors and tumors from ApcMin/+ mice, the questionable MCC gene was disrupted in human tumors but appeared to be intact in mouse tumors. CONCLUSIONS: These results indicate that MCC may not actually play any causative role in early colorectal tumorigenesis. We also hypothesize that its disruption in human CRCs is likely a mere result of its close proximity to APC in the human genome. Expanding this pilot study to the entire genome may identify more questionable genes like MCC, facilitating the discovery of new CRC driver gene candidates.

Mutations in DCC cause congenital mirror movements.

Mirror movements are involuntary contralateral movements that mirror voluntary ones and are often associated with defects in midline crossing of the developing central nervous system. We studied two large families, one French Canadian and one Iranian, in which isolated congenital mirror movements were inherited as an autosomal dominant trait. We found that affected individuals carried protein-truncating mutations in DCC (deleted in colorectal carcinoma), a gene on chromosome 18q21.2 that encodes a receptor for netrin-1, a diffusible protein that helps guide axon growth across the midline. Functional analysis of the mutant DCC protein from the French Canadian family revealed a defect in netrin-1 binding. Thus, DCC has an important role in lateralization of the human nervous system.

Methylation of the DCC gene is lost in advanced gastric cancer.

BACKGROUND: Deleted in colorectal carcinoma (DCC), one of the Netrin-1 receptors, belongs to the functional dependence receptor family, members of which share the ability to induce apoptosis in the absence of their ligands. Recently, we examined the methylation status of the DCC gene in colorectal carcinomas and found that aberrant methylation of the DCC gene was detected in 28 out of the 50 (56%) primary colon carcinomas. This result prompted us to examine the methylation status of the DCC gene in gastric carcinoma. MATERIALS AND METHODS: The methylation status of the DCC gene was examined in primary carcinomas and the corresponding normal tissues derived from 36 patients with gastric cancer using quantitative methylation-specific polymerase chain reaction (qMSP) and the correlation between the methylation status and the clinicopathological findings was evaluated. RESULTS: Aberrant methylation of the DCC gene was detected in 16 out of the 36 (44%) primary gastric carcinomas. A significant difference was observed in regard to the TNM stage (p=0.0093). CONCLUSION: DCC methylation was observed in the course of gastric carcinogenesis and disappeared in advanced gastric carcinoma.

Netrin and DCC: axon guidance regulators at the intersection of nervous system development and cancer.

In recent years, a number of axon guidance genes, including Netrin (Net) and Deleted in Colorectal Cancer (DCC), have been implicated in human cancers. Many of the hallmarks of human cancer, such as cell growth, invasion, evasion of apoptosis, and formation of a blood supply to the tumor, involve cellular processes that are critical during nervous system development. Here, the roles of Net-DCC in the regulation of these cellular processes in tumors and developing neurons are discussed. The advantages of using Drosophila to study the function of Net-DCC and other axon guidance molecules in these cellular processes, as well as the potential for cancer therapeutics targeting Net-DCC are highlighted.