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.

Genome-wide association study reveals novel genes for the ear size in sheep (Ovis aries).

Variations in ear size can be observed in livestock such as sheep; however, the genetic basis of variable ear size in sheep is still poorly understood. To investigate causative genes associated with ear size in sheep, a genome-wide association study was performed in 115 adult Duolang sheep with different-sized floppy ears using the Ovine Infinium HD BeadChip. We found 38 significant SNPs at the genome-wide or chromosome-wise 5% significance level after Bonferroni correction. The most significant association (P = 1.61 × 10-6 ) was found at SNP rs402740419, located in the DCC gene, which plays a critical role in ear development. Also, we observed two additional significant SNPs, rs407891215 in PTPRD and rs407769095 in SOX5, both of which are functionally associated with ear developmental processes. Our results are useful for future sheep breeding and provide insights into the genetic basis of ear size development in sheep and other livestock.

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).

Evolution of the functionally conserved DCC gene in birds.

Understanding the loss of conserved genes is critical for determining how phenotypic diversity is generated. Here we focus on the evolution of DCC, a gene that encodes a highly conserved neural guidance receptor. Disruption of DCC in animal models and humans results in major neurodevelopmental defects including commissural axon defects. Here we examine DCC evolution in birds, which is of particular interest as a major model system in neurodevelopmental research. We found the DCC containing locus was disrupted several times during evolution, resulting in both gene losses and faster evolution rate of salvaged genes. These data suggest that DCC had been lost independently twice during bird evolution, including in chicken and zebra finch, whereas it was preserved in many other closely related bird species, including ducks. Strikingly, we observed that commissural axon trajectory appeared similar regardless of whether DCC could be detected or not. We conclude that the DCC locus is susceptible to genomic instability leading to independent disruptions in different branches of birds and a significant influence on evolution rate. Overall, the phenomenon of loss or molecular evolution of a highly conserved gene without apparent phenotype change is of conceptual importance for understanding molecular evolution of key biological processes.

Using an Inbred Horse Breed in a High Density Genome-Wide Scan for Genetic Risk Factors of Insect Bite Hypersensitivity (IBH).

While susceptibility to hypersensitive reactions is a common problem amongst humans and animals alike, the population structure of certain animal species and breeds provides a more advantageous route to better understanding the biology underpinning these conditions. The current study uses Exmoor ponies, a highly inbred breed of horse known to frequently suffer from insect bite hypersensitivity, to identify genomic regions associated with a type I and type IV hypersensitive reaction. A total of 110 cases and 170 controls were genotyped on the 670K Axiom Equine Genotyping Array. Quality control resulted in 452,457 SNPs and 268 individuals being tested for association. Genome-wide association analyses were performed using the GenABEL package in R and resulted in the identification of two regions of interest on Chromosome 8. The first region contained the most significant SNP identified, which was located in an intron of the DCC netrin 1 receptor gene. The second region identified contained multiple top SNPs and encompassed the PIGN, KIAA1468, TNFRSF11A, ZCCHC2, and PHLPP1 genes. Although additional studies will be needed to validate the importance of these regions in horses and the relevance of these regions in other species, the knowledge gained from the current study has the potential to be a step forward in unraveling the complex nature of hypersensitive reactions.

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.

Mirror movement-like defects in startle behavior of zebrafish dcc mutants are caused by aberrant midline guidance of identified descending hindbrain neurons.

Mirror movements are involuntary movements on one side of the body that occur simultaneously with intentional movements on the contralateral side. Humans with heterozygous mutations in the axon guidance receptor DCC display such mirror movements, where unilateral stimulation results in inappropriate bilateral motor output. Currently, it is unclear whether mirror movements are caused by incomplete midline crossing and reduced commissural connectivity of DCC-dependent descending pathways or by aberrant ectopic ipsilateral axonal projections of normally commissural neurons. Here, we show that in response to unilateral tactile stimuli, zebrafish dcc mutant larvae perform involuntary turns on the inappropriate body side. We show that these mirror movement-like deficits are associated with axonal guidance defects of two identified groups of commissural reticulospinal hindbrain neurons. Moreover, we demonstrate that in dcc mutants, axons of these identified neurons frequently fail to cross the midline and instead project ipsilaterally. Whereas laser ablation of these neurons in wild-type animals does not affect turning movements, their ablation in dcc mutants restores turning movements. Thus, our results demonstrate that in dcc mutants, turns on the inappropriate side of the body are caused by aberrant ipsilateral axonal projections, and suggest that aberrant ipsilateral connectivity of a very small number of descending axons is sufficient to induce incorrect movement patterns.

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.

Preliminary screening for microsatellite instability and loss of heterozygosity in the deleted in colorectal cancer (DCC) gene among Filipino patients with colorectal adenocarcinoma

OBJECTIVE: This study aimed to detect the presence of microsatellite (MSI) and loss of heterozygosity (LOH) of the Deleted in Colorectal Cancer (DCC) gene in normal and tumor tissues of Filipino colorectal cancer patients and examine its correlation with age, gender, tumor grade, tumor stage and site of lesion.METHODS: Paired frozen normal and tumor tissues from thirtynine (39) patients with colorectal adenocarcinoma were used by polymerase chain reaction (PCR). Single strand conformation polymorphism - polyacrylamide gel electrophoresis (SSCP - PAGE) was used to determine MSI and restriction fragment length polymorphism (RFLP) was used to study LOH.RESULTS: Based on our data, out of the 39 patients, 10 showed LOH of the DCC gene using the LOH markers VNTR, M2 and M3, while no MSI was detected in the samples using the MSI markers BAT25 and BAT26. Correlation with clinicopathological characteristics showed that there is significance for the site of lesion. The LOH has correclation with tumor samples from the colon but not with those from the rectum.CONCLUSION: Preliminary screening for MSI and LOH of the DCC gene shows that occurrences of colorectal cancer among Filipino patients can be correlated with LOH of the DCC gene with colorectal cancer in a Filipino sample population.

dcc orchestrates the development of the prefrontal cortex during adolescence and is altered in psychiatric patients.

Adolescence is a period of heightened susceptibility to psychiatric disorders of medial prefrontal cortex (mPFC) dysfunction and cognitive impairment. mPFC dopamine (DA) projections reach maturity only in early adulthood, when their control over cognition becomes fully functional. The mechanisms governing this protracted and unique development are unknown. Here we identify dcc as the first DA neuron gene to regulate mPFC connectivity during adolescence and dissect the mechanisms involved. Reduction or loss of dcc from DA neurons by Cre-lox recombination increased mPFC DA innervation. Underlying this was the presence of ectopic DA fibers that normally innervate non-cortical targets. Altered DA input changed the anatomy and electrophysiology of mPFC circuits, leading to enhanced cognitive flexibility. All phenotypes only emerged in adulthood. Using viral Cre, we demonstrated that dcc organizes mPFC wiring specifically during adolescence. Variations in DCC may determine differential predisposition to mPFC disorders in humans. Indeed, DCC expression is elevated in brains of antidepressant-free subjects who committed suicide.

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.