BACKGROUND

Brain magnetic resonance imaging (MRI) and cognitive tests can identify heritable endophenotypes associated with an increased risk of developing stroke, dementia and Alzheimer's disease (AD). We conducted a genome-wide association (GWA) and linkage analysis exploring the genetic basis of these endophenotypes in a community-based sample.

METHODS

A total of 705 stroke- and dementia-free Framingham participants (age 62 +9 yrs, 50% male) who underwent volumetric brain MRI and cognitive testing (1999-2002) were genotyped. We used linear models adjusting for first degree relationships via generalized estimating equations (GEE) and family based association tests (FBAT) in additive models to relate qualifying single nucleotide polymorphisms (SNPs, 70,987 autosomal on Affymetrix 100K Human Gene Chip with minor allele frequency>> or = 0.10, genotypic call rate>> or = 0.80, and Hardy-Weinberg equilibrium p-value>> or = 0.001) to multivariable-adjusted residuals of 9 MRI measures including total cerebral brain (TCBV), lobar, ventricular and white matter hyperintensity (WMH) volumes, and 6 cognitive factors/tests assessing verbal and visuospatial memory, visual scanning and motor speed, reading, abstract reasoning and naming. We determined multipoint identity-by-descent utilizing 10,592 informative SNPs and 613 short tandem repeats and used variance component analyses to compute LOD scores.

RESULTS

The strongest gene-phenotype association in FBAT analyses was between SORL1 (rs1131497; p = 3.2 x 10(-6)) and abstract reasoning, and in GEE analyses between CDH4 (rs1970546; p = 3.7 x 10(-8)) and TCBV. SORL1 plays a role in amyloid precursor protein processing and has been associated with the risk of AD. Among the 50 strongest associations (25 each by GEE and FBAT) were other biologically interesting genes. Polymorphisms within 28 of 163 candidate genes for stroke, AD and memory impairment were associated with the endophenotypes studied at p < 0.001. We confirmed our previously reported linkage of WMH on chromosome 4 and describe linkage of reading performance to a marker on chromosome 18 (GATA11A06), previously linked to dyslexia (LOD scores = 2.2 and 5.1).

CONCLUSIONS

Our results suggest that genes associated with clinical neurological disease also have detectable effects on subclinical phenotypes. These hypothesis generating data illustrate the use of an unbiased approach to discover novel pathways that may be involved in brain aging, and could be used to replicate observations made in other studies.

OBJECTIVE

To further understand the molecular pathogenesis of pulmonary sarcomatoid carcinoma (PSC) and develop new therapeutic strategies in this treatment-refractory disease.

METHODS

Whole-exome sequencing in a discovery set (n = 10) as well as targeted MET mutation screening in an independent validation set (n = 26) of PSC were performed. Reverse transcriptase polymerase chain reaction and Western blotting were performed to validate MET exon 14 skipping. Functional studies for validation of the oncogenic roles of MET exon 14 skipping were conducted in lung adenosquamous cell line H596 (MET exon 14 skipped and PIK3CA mutated) and gastric adenocarcinoma cell line Hs746T (MET exon 14 skipped). Response to MET inhibitor therapy with crizotinib in a patient with advanced PSC and MET exon 14 skipping was evaluated to assess clinical translatability.

RESULTS

In addition to confirming mutations in known cancer-associated genes (TP53, KRAS, PIK3CA, MET, NOTCH, STK11, and RB1), several novel mutations in additional genes, including RASA1, CDH4, CDH7, LAMB4, SCAF1, and LMTK2, were identified and validated. MET mutations leading to exon 14 skipping were identified in eight (22%) of 36 patient cases; one of these tumors also harbored a concurrent PIK3CA mutation. Short interfering RNA silencing of MET and MET inhibition with crizotinib showed marked effects on cell viability and decrease in downstream AKT and mitogen-activated protein kinase activation in Hs746T and H596 cells. Concurrent PIK3CA mutation required addition of a second agent for successful pathway suppression and cell viability effect. Dramatic response to crizotinib was noted in a patient with advanced chemotherapy-refractory PSC carrying a MET exon 14 skipping mutation.

CONCLUSIONS

Mutational events of MET leading to exon 14 skipping are frequent and potentially targetable events in PSC.

Gene promoter methylation causes loss of tumor suppressor genes function in human cancer. Here, we show that the CDH4 gene, a member of the cadherin family encoding for R-cadherin, contains a CpG island located at the 5' of the first exon, which functions as a promoter element and is frequently affected by methylation in human cancer. By using methylation-specific PCR and reverse transcription-PCR in human cancer cell lines, promoter methylation could be directly linked to loss of gene expression. After treatment with the demethylating agent 5-aza-2-deoxycytidine, expression could be restored. Analysis of human primary tumors revealed that the CDH4 gene is methylated in 78% (38 of 49) of colorectal and 95% (20 of 21) of gastric carcinomas. CDH4 methylation was not detected in nonneoplastic colonic (0 of 10) and stomach (0 of 10) tissues or in peripheral blood (0 of 17). CDH4 methylation was detected in histologically normal tissues located in proximity of the neoplasms, indicating that CDH4 methylation is an early event in gastrointestinal tumor progression. We also proved that CDH4 methylation can be revealed in the peripheral blood of cancer patients. Our results indicate that CDH4 may act as a tumor suppressor gene in human gastrointestinal tumors and can potentially be used as an early diagnostic marker for gastrointestinal tumorigenesis.

Epigenetic changes play significant roles in the development of cancer. UHRF1, as an epigenetic regulator, has been shown to be overexpressed and to coordinate tumor suppressor gene silencing in several cancers. However, the role and underlying mechanism of UHRF1 in gastric cancer (GC) progression remain largely unknown. In this study, we investigated the expression and function of UHRF1 in GC metastasis and explored its upstream regulatory mechanisms at the microRNA level. UHRF1 was overexpressed in GC tissues, especially in metastatic ones, and a high level of UHRF1 expression predicted poor survival. The down-regulation of UHRF1 suppressed GC invasion and metastasis in vitro and in vivo. We identified and verified miR-146a and miR-146b as direct upstream regulators of UHRF1. Furthermore, the restoration of miR-146a/b dramatically reduced the expression of UHRF1 through the direct targeting of its 3'-UTR, and this effect in turn reactivated the slit homologue 3 (Slit3), cadherin 4 (CDH4), and runt-related transcription factor 3 (RUNX3) genes via promoter demethylation. Finally, analyses of miR-146a/b and UHRF1 levels in human GC tissues revealed that miR-146a/b correlated inversely with UHRF1 expression. These findings describe a new mechanism for the regulation of UHRF1 and aberrant DNA hypermethylation in GC. The newly identified miR-146a/b/UHRF1 axis provides insight into the GC metastasis process, and targeting this novel axis represents a therapeutic approach to blocking GC metastasis.

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. In this study, we aimed to evaluate to the utility of aberrant promoter hypermethylation for detection in a panel of 10 genes (KIF1A, EDNRB, CDH4, TERT, CD44, NISCH, PAK3, VGF, MAL and FKBP4) in head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. We investigated methylation of the gene promoters by bisulfite modification and quantitative methylation-specific PCR (Q-MSP) in a preliminary study of a limited cohort of salivary rinses from healthy subjects (n = 61) and patients with HNSCC (n = 33). The methylation status of 2 selected genes (EDNRB and KIF1A) were then analyzed in 15 normal mucosa samples from a healthy population, 101 HNSCC tumors and the corresponding salivary rinses from 71 out of the 101 HNSCC patients were collected before treatment. The promoter regions of CDH4, TERT, VGF, MAL, FKBP4, NISCH and PAK3 were methylated in normal salivary rinses while no methylation of CD44 was observed in either normal salivary rinses or tumor samples. However, KIF1A and EDNRB were methylated in 98 and 97% of primary HNSCC tissues respectively and were only methylated in 2 and 6.6% of normal salivary rinses. In addition, KIF1A and EDNRB were methylated in 38 and 67.6% of salivary rinses from HNSCC patients, respectively. Promoter hypermethylation of KIF1A and EDNRB is a frequent event in primary HNSCC, and these genes are preferentially methylated in salivary rinses from HNSCC patients. KIF1A and EDNRB are potential biomarkers for HNSCC detection.

The transcription factor Pax6 has been implicated in two processes that may be related in brain development: establishment of regional cell adhesion properties and axon guidance. In Pax6 mutant mouse embryos, forebrain pioneer axons make pathfinding errors. These errors occur in a region of the ventral thalamus in which the expression of the cell adhesion molecule R-cadherin (Cdh4) is lost in Pax6 mutants. In vitro, an R-cadherin substrate promoted pioneer axon outgrowth. Furthermore, pioneer axon outgrowth was rescued in vivo by selective replacement of R-cadherin by electroporation into cultured Pax6 mutant embryos. Thus, these studies implicate Pax6 as an early brain patterning gene that establishes regional adhesive codes to guide pioneer axons.

In zebrafish, R-cadherin (cadherin-4 or Cdh4) is expressed in the retina and in retinorecipient brain regions, suggesting that Cdh4 functions during visual system development. Cdh4 function was examined during retinogenesis and retinal axon outgrowth using antisense morpholino oligonucleotides and mutant Cdh4 construct expression. In knockdowns, Cdh4 was reduced or absent, eyes were small, and retinae lacked discrete laminae. Increased cell death produced the small eye phenotype. Zn5-, Pax6-, and zpr-1-positive cells were reduced or absent in knockdown retinas but, when present, were in the correct laminae. Cdh4 knockdowns had sparse or absent retinal ganglion cell axons. When present, axons projected contralaterally but lacked fine branching and failed to reach the tectum or arborize the entire tectum. Mutant Cdh4 construct expression during retinal ganglion cell differentiation reduced or ablated neurite formation. Cdh4 is necessary for neural retina survival and differentiation, and required for normal retinotectal projection formation and tectal arborization.

We investigated the transcription levels, promoter methylation status and role as a tumor suppressor gene (TSG) of the cadherin CDH4 in nasopharyngeal carcinoma (NPC). The expression of CDH4 was decreased in NPC cell lines, xenografts and primary tumor biopsies. Promoter hypermethylation of CDH4 was detected in all five NPC cell lines, both NPC xenograft lines and 94.3% of primary tumors but not in any of the 12 normal epithelial samples. Loss of CDH4 expression could be restored by the methyltransferase inhibitor 5-aza-2'-deoxycytidine in NPC cell lines. Ectopic expression of CDH4 in the NPC cell lines inhibits cell proliferation, colony formation, migration and elicit cell communication. CDH4 may be a novel putative TSG that can be frequently and tumor-specifically inactivated by its promoter methylation in NPC.

To clarify the structural basis of the cell adhesion activity of cadherins, we examined the effects of point mutations of well-conserved amino acid residues in the extracellular domain 1 of cadherin-4 (Cdh4) on the adhesion properties by alanine scanning mutagenesis. Mutations of two well-conserved aromatic amino acid residues in the extracellular domain 1 resulted in abnormal processing of Cdh4 molecules and no cell adhesion activity, whereas mutations of the corresponding aromatic amino acids in the extracellular domain 2 did not show these effects, suggesting a role for the two residues in the extracellular domain 1 in the folding and/or intracellular transport processes of Cdh4. Mutations of the amino acid residues suspected to be involved in strand dimer formation resulted in loss or significant decrease in cell adhesion activity. The mutant Cdh4s showed weak concentration at cell-cell adhesion sites and chemical cross-linking suggested that the strand dimer formation was actually impaired in the mutants. These results are consistent with the zipper model, in which the extracellular domain 1 of Cdh4 has intrinsic strand dimer formation activity in addition to adhesion dimer formation activity, both of which are involved in cell adhesion activity. The zipper model, however, needs further improvement to fully account for the present results.

Cadherin superfamily genes play a role in a wide variety of developmental processes and mature functions of the vertebrate brain. In the present study, we mapped in situ the expression pattern of five classic cadherins (Cdh4, Cdh6, Cdh7, Cdh8, Cdh11) and eight δ-protocadherins (Pcdh1, Pcdh7, Pcdh8, Pcdh9, Pcdh10, Pcdh11, Pcdh17 and Pcdh19) in the primary somatosensory cortex of the adult mouse. All of these cadherins show layer-specific expression profiles in primary somatosensory cortex. Some cadherins (for example, Cdh4, Cdh7, Pcdh8) mark subsets of cells within a given lamina, while other cadherins (Cdh11 and Pcdh10) are expressed more widely in multiple layers. Results from tyramide-based double-fluorescence in situ hybridization (FISH) provide evidence that most single neurons express more than one cadherin in a combinatorial fashion in all layers of cerebral cortex. This combinatorial code is rather comprehensive because pairwise expression of cadherins can assume any type of combination (complementarity, partial or complete overlap, subset-specific expression, cell-size specific expression, etc.). We propose that the combinatorial expression of multiple cadherin genes contributes to the molecular specification of the vast complexity of neurons in cerebral cortex.

Cadherins are homophilic cell adhesion molecules that control development of a variety of tissues and maintenance of adult structures. Although cadherins have been implicated in the development of the brain, including the visual system, in several vertebrate species, little is known of their role in zebrafish. In this study, we examined distribution of cadherin-2 (Cdh2, N-cadherin) in the visual system of developing and adult zebrafish using both immunocytochemical and in situ hybridization methods, and we compared Cdh2 distribution to that of the previously reported and closely related cadherin-4 (Cdh4, R-cadherin). As in other vertebrates, in zebrafish embryos Cdh2 was widely expressed in the early nervous system, but its expression became more restricted as development proceeded. Cdh4 was not detectable until later in development, at about the time when the first ganglion cells are generated. Cdh2 and Cdh4 were expressed in distinct regions of developing visual structures, including the lens. We hypothesize that the differential expression of these two cadherins in developing zebrafish visual structures reflects functionally different roles in the development of the vertebrate visual system.

Cadherins are a superfamily of Ca(2+)-dependent cell surface glycoproteins that play a morphogenetic role in a wide variety of developmental processes. They provide a code of potentially adhesive cues for layer formation in mammalian cerebral cortex. One of the animal models used for studying corticogenesis is the reeler mouse. Previous investigations showed that radial neuronal migration is impaired in this mutant, possibly resulting in an inversion of cortical layers. However, the extent of this "outside-in" cortical layering remains unclear. In the present study, we investigated the mRNA expression of cadherins (Cdh4, Cdh6, Cdh7, Cdh8, Pcdh8, Pcdh9, Pcdh11, Pcdh17, and Pcdh19) in the cerebral cortex of wild-type (wt) mice and reeler mutants. All cadherins show a layer-specific expression profile in wt mice, but, in reeler cortex, cadherin-expressing cells are distributed widely across the radial dimension. The altered layering in reeler mutants completely disrupts the radial expression of cadherins, which is more patchy, rather than laminar. Regionalized gradient-like expression of cadherins is preserved. Our findings are compatible with a model, in which the ubiquitous dispersion of cadherin-expressing cells results from a dysgenesis of radial glial cells and a misrouting of migrating neuroblasts.

OBJECTIVE

Dexamethasone (DEX) is a glucocorticoid commonly used in topical eyedrops to treat eye inflammation. It has an undesirable effect of inducing glaucoma in certain patients. In human Trabecular Meshwork (TM) cells DEX regulates a number of genes but its global influence on TM gene expression is still elusive. In the present work, DEX effects on global gene expressions of an established human TM cell line were studied by microarray.

METHODS

The whole experiment of microarray was repeated three times. Differentially expressed genes were identified by an empirical Bayes approach and confirmed by Reverse Transcription Polymerase Chain Reaction.

RESULTS

Eight genes (GAS1, CDH4, MT1L, CST3, ATF4, ASNS/TS11, CHOP, HSPA5) were identified that are at least a thousand times more likely to be differentially expressed due to DEX treatment and six genes (TSC22, LDHA, IGFBP2, TAGLN, SCG2, WARS) were identified that are at least a hundred times more likely to be differentially expressed due to DEX treatment. Except for MT1L, ASNS/TS11, IGFBP2, SCG2, and WARS, all the other genes are first reported here to be regulated by DEX in TM. Intriguingly, several of them have overlapping roles in anti-inflammatory response and outflow resistance.

CONCLUSIONS

The results of our experiments on cultured human TM cells indicate that the increase in outflow resistance and ultimate ocular hypertension may be byproducts of the favorable anti-inflammatory response triggered by DEX.

Intake of anthocyanin-rich foods has been associated with a reduced risk of cardiovascular diseases. We recently reported that a nutritional supplementation with a bilberry anthocyanin-rich extract (BE) attenuates atherosclerotic lesion development in apolipoprotein E-deficient (apoE⁻/⁻) mice. However, the mechanism(s) of their preventive action are not completely understood. Anthocyanins may alter mRNA levels of genes related to atherosclerosis in cultured macrophages and endothelial cells, but in vivo studies remain scarce. The aim of the present study was to explore the in vivo mechanisms of action of the same bilberry extract, administered by supplementation at a nutritional level, in the aorta of apo E⁻/⁻ mice using a global transcriptomic approach. This study focused on the early stage of atherosclerosis development for better assessment of BE action on initiation mechanisms of this pathology. After a two week period, plasma lipid and antioxidant capacity were evaluated and the global genomic analysis was carried out using pangenomic microarrays. BE supplementation significantly improved hypercholesterolemia whereas the plasmatic antioxidant status remained unchanged. Nutrigenomic analysis identified 1261 genes which expression was modulated by BE in the aorta. Bioinformatic analysis revealed that these genes are implicated in different cellular processes such as oxidative stress, inflammation, transendothelial migration and angiogenesis, processes associated with atherosclerosis development/protection. Some of the most significantly down-regulated genes included genes coding for AOX1, CYP2E1 or TXNIP implicated in the regulation of oxidative stress, JAM-A coding for adhesion molecules or VEGFR2 implicate in regulation of angiogenesis. Other genes were up-regulated, such as CRB3, CLDN14 or CDH4 potentially associated with increased cell-cell adhesion and decreased paracellular permeability. These results provide a global integrated view of the mechanisms involved in the preventive action of bilberry anthocyanin-rich extract against atherosclerosis.

The amygdaloid complex represents a group of telencephalic nuclei and cortical areas that control emotional and social behavior. Amygdalar development is poorly understood. It is generally accepted that the structures of the amygdala originate from the neuroepithelium at both sides of the pallial-subpallial boundary. In the present study, we mapped the expression of 13 members of the cadherin superfamily of cell adhesion molecules, which provide an adhesive code for the development and maintenance of functional structures in the central nervous system (CNS). Five classic cadherins (Cdh4, Cdh6, Cdh7, Cdh8, Cdh11) and eight delta-protocadherins (Pcdh1, Pcdh7, Pcdh8, Pcdh9, Pcdh10, Pcdh11, PCdh17, PCdh19) were studied by in situ hybridization in the postnatal (P5) and adult mouse amygdala. In the different parts of the amygdala, each of these (proto-) cadherins shows a distinct and spatially restricted expression pattern that is highly similar at postnatal and adult stages. The combinatorial expression of (proto-) cadherins allows the distinction of multiple molecular subdivisions within the amygdala that partially coincide with previously described morphological divisions. Beyond these expected results, a number of novel molecular subdivisions and subpopulations of cells were identified; for example, additional molecular subdomains, patches, or cell aggregates with distinct (proto-) cadherin expression in several nuclei/areas of the amygdala. We also show that several cadherins are molecular markers for particular functional subsystems within the amygdala, such as in the olfactory projections. In summary, (proto-) cadherins provide a code of potentially adhesive cues that can aid the understanding of functional organization in the amygdala.

The intrinsic drivers of migration in glioblastoma (GBM) are poorly understood. To better capture the native molecular imprint of GBM and its developmental context, here we isolate human stem cell populations from GBM (GSC) and germinal matrix tissues and map their chromatin accessibility via ATAC-seq. We uncover two distinct regulatory GSC signatures, a developmentally shared/proliferative and a tumor-specific/migratory one in which TEAD1/4 motifs are uniquely overrepresented. Using ChIP-PCR, we validate TEAD1 trans occupancy at accessibility sites within AQP4, EGFR, and CDH4. To further characterize TEAD's functional role in GBM, we knockout TEAD1 or TEAD4 in patient-derived GBM lines using CRISPR-Cas9. TEAD1 ablation robustly diminishes migration, both in vitro and in vivo, and alters migratory and EMT transcriptome signatures with consistent downregulation of its target AQP4. TEAD1 overexpression restores AQP4 expression, and both TEAD1 and AQP4 overexpression rescue migratory deficits in TEAD1-knockout cells, implicating a direct regulatory role for TEAD1-AQP4 in GBM migration.

We previously reported that cadherin-4 (also called R-cadherin) was expressed by the majority of the developing zebrafish cranial and lateral line ganglia. Cadherin-4 (Cdh4) function in the formation of these structures in zebrafish was studied using morpholino antisense technology. Differentiation of the cranial and lateral line ganglia and lateral line nerve and neuromasts of the cdh4 morphants was analyzed using multiple neural markers. We found that a subset of the morphant cranial and lateral line ganglia were disorganized, smaller, with reduced staining, and/or with altered shape compared to control embryos. Increased cell death in the morphant ganglia likely contributed to these defects. Moreover, cdh4 morphants had shorter lateral line nerves and a reduced number of neuromasts, which was likely caused by disrupted migration of the lateral line primordia. These results indicate that Cdh4 plays a role in the normal formation of the zebrafish lateral line system and a subset of the cranial ganglia.

Although odor receptors have been implicated in establishing the topography of olfactory sensory neurons (OSNs) in the olfactory bulb (OB), it is likely other molecules are also involved. The cadherins (CDHs) are a large family of cell adhesion molecules that mediate cell:cell interactions elsewhere in the central nervous system. However, their distribution and role in the olfactory system have remained largely unexplored. We previously demonstrated that intracellular binding partners of cadherins, the catenins, have unique spatiotemporal patterns of expression in the developing olfactory system. To further our understanding of cadherin function within the developing olfactory system, we now report on the localization of 11 classical cadherins-CDH1, 2, 3, 4, 5, 6, 8, 10, 11, 13, and 15. We demonstrate the expression of all but CDH5 and CDH15 in neuronal and/or glial cells in primary olfactory structures. CDH1 and CDH2 are expressed by OSNs; CDH2 expression closely parallels that seen for gamma-catenin in OSN axons. CDH3 and CDH11 are expressed by olfactory ensheathing glia, which surround OSN axons in the outer OB. CDH2, CDH4, and CDH6 are expressed within neuropil. CDH2, CDH4, CDH6, CDH8, CDH10, CDH11, and CDH13 are expressed by projection neurons within the main and accessory OBs. We conclude that cadherin proteins in the developing olfactory system are positioned to underlie the formation of the odorant map and local circuits within the OB.