Preliminary report: Late seizure recurrence years after epilepsy surgery may be associated with alterations in brain tissue transcriptome.

We recently proposed that the maturation of a new epileptic focus (epileptogenesis) may explain late seizure recurrences, starting months to years after resective epilepsy surgery. We explore here the hypothesis that inherent transcriptomic changes may distinguish such "late relapsers." An in-depth clinical review of 2 patients with recurrent seizures starting years after surgery is contrasted to 4 controls who remained seizure-free postoperatively. This clinical analysis is combined with RNA sequencing from the resected brain tissue, followed by unsupervised hierarchical clustering, independent pathway analysis, and multidimensional scaling analysis. Late-recurrence patients clustered apart from seizure-free patients, with late recurrence patients clustering together in the central space, whereas the seizure-free patients clustered together in the periphery. We utilized RNA-seq to identify differentially expressed genes between late-recurrence and seizure-free samples. We found 29 annotated genes with statistically significant differential expression (q < 0.05). The top canonical pathways identified as distinctly separating the late-recurrence patients from the seizure-free patients included the intrinsic prothrombin activation pathway (p = 1.55E-06), the complement system (p = 4.57E-05), and the atherosclerosis signaling pathway (p = 4.57E-05). Our observations suggest that late recurrences after epilepsy surgery may be influenced partly by differences in gene expression in neuroinflammatory and brain healing/remodeling pathways. Such a hypothesis needs to be validated in the future.

Unexpected cancer-predisposition gene variants in Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome patients without underlying germline PTEN mutations.

Patients with heritable cancer syndromes characterized by germline PTEN mutations (termed PTEN hamartoma tumor syndrome, PHTS) benefit from PTEN-enabled cancer risk assessment and clinical management. PTEN-wildtype patients (~50%) remain at increased risk of developing certain cancers. Existence of germline mutations in other known cancer susceptibility genes has not been explored in these patients, with implications for different medical management. We conducted a 4-year multicenter prospective study of incident patients with features of Cowden/Cowden-like (CS/CS-like) and Bannayan-Riley-Ruvalcaba syndromes (BRRS) without PTEN mutations. Exome sequencing and targeted analysis were performed including 59 clinically actionable genes from the American College of Medical Genetics and Genomics (ACMG) and 24 additional genes associated with inherited cancer syndromes. Pathogenic or likely pathogenic cancer susceptibility gene alterations were found in 7 of the 87 (8%) CS/CS-like and BRRS patients and included MUTYH, RET, TSC2, BRCA1, BRCA2, ERCC2 and HRAS. We found classic phenotypes associated with the identified genes in 5 of the 7 (71.4%) patients. Variant positive patients were enriched for the presence of second malignant neoplasms compared to patients without identified variants (OR = 6.101, 95% CI 1.143-35.98, p = 0.035). Germline variant spectrum and frequencies were compared to The Cancer Genome Atlas (TCGA), including 6 apparently sporadic cancers associated with PHTS. With comparable overall prevalence of germline variants, the spectrum of mutated genes was different in our patients compared to TCGA. Intriguingly, we also found notable enrichment of variants of uncertain significance (VUS) in our patients (OR = 2.3, 95% CI 1.5-3.5, p = 0.0002). Our data suggest that only a small subset of PTEN-wildtype CS/CS-like and BRRS patients could be accounted for by germline variants in some of the known cancer-related genes. Thus, the existence of alterations in other and more likely non-classic cancer-associated genes is plausible, reflecting the complexity of these heterogeneous hereditary cancer syndromes.

Breast tissue, oral and urinary microbiomes in breast cancer.

It has long been proposed that the gut microbiome contributes to breast carcinogenesis by modifying systemic estrogen levels. This is often cited as a possible mechanism linking breast cancer and high-fat, low-fiber diets as well as antibiotic exposure, associations previously identified in population-based studies. More recently, a distinct microbiome has been identified within breast milk and tissue, but few studies have characterized differences in the breast tissue microbiota of patients with and without cancer, and none have investigated distant body-site microbiomes outside of the gut. We hypothesize that cancerous breast tissue is associated with a microbiomic profile distinct from that of benign breast tissue, and that microbiomes of more distant sites, the oral cavity and urinary tract, will reflect dysbiosis as well. Fifty-seven women with invasive breast cancer undergoing mastectomy and 21 healthy women undergoing cosmetic breast surgery were enrolled. The bacterial 16S rRNA gene was amplified from urine, oral rinse and surgically collected breast tissue, sequenced, and processed through a QIIME-based bioinformatics pipeline. Cancer patient breast tissue microbiomes clustered significantly differently from non-cancer patients (p=0.03), largely driven by decreased relative abundance of Methylobacterium in cancer patients (median 0.10 vs. 0.24, p=0.03). There were no significant differences in oral rinse samples. Differences in urinary microbiomes were largely explained by menopausal status, with peri/postmenopausal women showing decreased levels of Lactobacillus. Independent of menopausal status, however, cancer patients had increased levels of gram-positive organisms including Corynebacterium (p<0.01), Staphylococcus (p=0.02), Actinomyces (p<0.01), and Propionibacteriaceae (p<0.01). Our observations suggest that the local breast microbiota differ in patients with and without breast cancer. Cancer patient urinary microbiomes were characterized by increased levels of gram-positive organisms in this study, but need to be further studied in larger cohorts.

Microbiomic differences in tumor and paired-normal tissue in head and neck squamous cell carcinomas.

BACKGROUND: While the role of the gut microbiome in inflammation and colorectal cancers has received much recent attention, there are few data to support an association between the oral microbiome and head and neck squamous cell carcinomas. Prior investigations have been limited to comparisons of microbiota obtained from surface swabs of the oral cavity. This study aims to identify microbiomic differences in paired tumor and non-tumor tissue samples in a large group of 121 patients with head and neck squamous cell carcinomas and correlate these differences with clinical-pathologic features. METHODS: Total DNA was extracted from paired normal and tumor resection specimens from 169 patients; 242 samples from 121 patients were included in the final analysis. Microbiomic content of each sample was determined using 16S rDNA amplicon sequencing. Bioinformatic analysis was performed using QIIME algorithms. F-testing on cluster strength, Wilcoxon signed-rank testing on differential relative abundances of paired tumor-normal samples, and Wilcoxon rank-sum testing on the association of T-stage with relative abundances were conducted in R. RESULTS: We observed no significant difference in measures of alpha diversity between tumor and normal tissue (Shannon index: p = 0.13, phylogenetic diversity: p = 0.42). Similarly, although we observed statistically significantly differences in both weighted (p = 0.01) and unweighted (p = 0.04) Unifrac distances between tissue types, the tumor/normal grouping explained only a small proportion of the overall variation in the samples (weighted R2 = 0.01, unweighted R2 < 0.01). Notably, however, when comparing the relative abundances of individual taxa between matched pairs of tumor and normal tissue, we observed that Actinomyces and its parent taxa up to the phylum level were significantly depleted in tumor relative to normal tissue (q < 0.01), while Parvimonas was increased in tumor relative to normal tissue (q = 0.01). These differences were more pronounced among patients with more extensive disease as measured by higher T-stage. CONCLUSIONS: Matched pairs analysis of individual tumor-normal pairs revealed significant differences in relative abundance of specific taxa, namely in the genus Actinomyces. These differences were more pronounced among patients with higher T-stage. Our observations suggest further experiments to interrogate potential novel mechanisms relevant to carcinogenesis associated with alterations of the oral microbiome that may have consequences for the human host.

Germline compound heterozygous poly-glutamine deletion in USF3 may be involved in predisposition to heritable and sporadic epithelial thyroid carcinoma.

Cowden syndrome (CS) is an autosomal dominant disorder that predisposes to breast, thyroid, and other epithelial cancers. Differentiated thyroid carcinoma (DTC), as one of the major component cancers of CS, is the fastest rising incident cancer in the USA, and the most familial of all solid tumours. To identify additional candidate genes of CS and potentially DTC, we analysed a multi-generation CS-like family with papillary thyroid cancer (PTC), applying a combined linkage-based and whole-genome sequencing strategy and identified an in-frame germline compound heterozygous deletion, p.[Gln1478del];[Gln1476-Gln1478del] in USF3 (previously known as KIAA2018). Among 90 unrelated CS/CS-like individuals, 29% were found to have p.[Gln1478del];[Gln1476-Gln1478del]. Of 497 TCGA PTC individuals, 138 (27%) were found to carry this germline compound deletion, with somatically decreased tumour USF3 expression. We demonstrate an increased migration phenotype along with enhanced epithelial-to-mesenchymal transition (EMT) signature after USF3 knockdown or USF3 p.[Gln1478del];[Gln1476-Gln1478del] overexpression, which sensitizes cells to the endoplasmic reticulum (ER) stress. Loss of USF3 function induced cell necrosis-like features and impaired respiratory capacity while providing a glutamine-dependent cell survival advantage, strongly suggests a metabolic survival and migration-favouring microenvironment for carcinogenesis. Therefore, USF3 may be involved in the predisposition of thyroid cancer. Importantly, the results that glutamine-dependent survival and sensitivity to ER stress in USF3-deficient cells provide avenues for therapeutic and adjunct preventive interventions for both sporadic cancer as well as cancer predisposition syndromes with similar mechanisms.

Exome sequencing reveals germline gain-of-function EGFR mutation in an adult with Lhermitte-Duclos disease.

Lhermitte-Duclos disease (LDD) is a rare cerebellar disorder believed to be pathognomonic for Cowden syndrome. Presently, the only known etiology is germline PTEN mutation. We report a 41-yr-old white female diagnosed with LDD and wild-type for PTEN. Exome sequencing revealed a germline heterozygous EGFR mutation that breaks a disulfide bond in the receptor's extracellular domain, resulting in constitutive activation. Functional studies demonstrate activation of ERK/AKT signaling pathways, mimicking PTEN loss-of-function downstream effects. The identification of EGFR as a candidate LDD susceptibility gene contributes to advancement of molecular diagnosis and targeted therapy for this rare condition with limited treatment options.

Cancer-predisposition gene KLLN maintains pericentric H3K9 trimethylation protecting genomic stability.

Maintenance of proper chromatin states and genomic stability is vital for normal development and health across a range of organisms. Here, we report on the role of KLLN in maintenance of pericentric H3K9 trimethylation (H3K9me3) and genomic stability. Germline hypermethylation of KLLN, a gene uncovered well after the human genome project, has been linked to Cowden cancer-predisposition syndrome (CS) in PTEN wild-type cases. KLLN first identified as a p53-dependent tumor suppressor gene, was believed to bind randomly to DNA and cause S-phase arrest. Using chromatin immunoprecipitation-based sequencing (ChIP-seq), we demonstrated that KLLN binds to DNA regions enriched with H3K9me3. KLLN overexpression correlated with increased H3K9 methyltransferase activity and increased global H3K9me3, while knockdown of KLLN had an opposite effect. We also found KLLN to localize to pericentric regions, with loss of KLLN resulting in dysregulation of pericentric heterochromatin, with consequent chromosomal instability manifested by increased micronuclei formation and numerical chromosomal aberrations. Interestingly, we show that KLLN interacts with DBC1, with consequent abrogation of DBC1 inhibition of SUV39H1, a H3K9 methyltransferase, suggesting the mode of KLLN regulating H3K9me3. These results suggest a critical role for KLLN as a potential regulator of pericentric heterochromatin formation, genomic stability and gene expression.

Germline Heterozygous Variants in SEC23B Are Associated with Cowden Syndrome and Enriched in Apparently Sporadic Thyroid Cancer.

Cancer-predisposing genes associated with inherited cancer syndromes help explain mechanisms of sporadic carcinogenesis and often inform normal development. Cowden syndrome (CS) is an autosomal-dominant disorder characterized by high lifetime risks of epithelial cancers, such that ∼50% of affected individuals are wild-type for known cancer-predisposing genes. Using whole-exome and Sanger sequencing of a multi-generation CS family affected by thyroid and other cancers, we identified a pathogenic missense heterozygous SEC23B variant (c.1781T>G [p.Val594Gly]) that segregates with the phenotype. We also found germline heterozygous SEC23B variants in 3/96 (3%) unrelated mutation-negative CS probands with thyroid cancer and in The Cancer Genome Atlas (TCGA), representing apparently sporadic cancers. We note that the TCGA thyroid cancer dataset is enriched with unique germline deleterious SEC23B variants associated with a significantly younger age of onset. SEC23B encodes Sec23 homolog B (S. cerevisiae), a component of coat protein complex II (COPII), which transports proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Interestingly, germline homozygous or compound-heterozygous SEC23B mutations cause an unrelated disorder, congenital dyserythropoietic anemia type II, and SEC23B-deficient mice suffer from secretory organ degeneration due to ER-stress-associated apoptosis. By characterizing the p.Val594Gly variant in a normal thyroid cell line, we show that it is a functional alteration that results in ER-stress-mediated cell-colony formation and survival, growth, and invasion, which reflect aspects of a cancer phenotype. Our findings suggest a different role for SEC23B, whereby germline heterozygous variants associate with cancer predisposition potentially mediated by ER stress "addiction."

Exome Sequencing Reveals Germline SMAD9 Mutation That Reduces Phosphatase and Tensin Homolog Expression and Is Associated With Hamartomatous Polyposis and Gastrointestinal Ganglioneuromas.

Hamartomatous polyposis syndromes (HPS) account for a small but appreciable proportion of inherited gastrointestinal cancer predisposition syndromes; patients with HPS have an increased risk for colon and extracolonic malignancies. We present a unique case of familial juvenile polyposis syndrome associated with gastrointestinal ganglioneuromas of unknown etiology. The patient was tested for HPS-associated genes, but no mutation was detected. Exome sequencing identified a germline heterozygous mutation in SMAD9 (SMAD9(V90M)). This mutation was predicted to be an activating mutation. HEK cells transfected to express SMAD9(V90M) had reduced expression of phosphatase and tensin homolog; this reduction was also observed in a polyp from the patient. We have therefore identified a new susceptibility locus for HPS. Patients with hamartomatous polyposis in the colon associated with ganglioneuromatosis should be referred for genetic assessments.

Negative regulation of the interferon response by an interferon-induced long non-coding RNA.

Long non-coding RNAs (lncRNAs) play critical roles in diverse cellular processes; however, their involvement in many critical aspects of the immune response including the interferon (IFN) response remains poorly understood. To address this gap, we compared the global gene expression pattern of primary human hepatocytes before and at three time points after treatment with IFN-α. Among ∼ 200 IFN-induced lncRNAs, one transcript showed ∼ 100-fold induction. This RNA, which we named lncRNA-CMPK2, was a spliced, polyadenylated nuclear transcript that was induced by IFN in diverse cell types from human and mouse. Similar to protein-coding IFN-stimulated genes (ISGs), its induction was dependent on JAK-STAT signaling. Intriguingly, knockdown of lncRNA-CMPK2 resulted in a marked reduction in HCV replication in IFN-stimulated hepatocytes, suggesting that it could affect the antiviral role of IFN. We could show that lncRNA-CMPK2 knockdown resulted in upregulation of several protein-coding antiviral ISGs. The observed upregulation was caused by an increase in both basal and IFN-stimulated transcription, consistent with loss of transcriptional inhibition in knockdown cells. These results indicate that the IFN response involves a lncRNA-mediated negative regulatory mechanism. lncRNA-CMPK2 was strongly upregulated in a subset of HCV-infected human livers, suggesting a role in modulation of the IFN response in vivo.

A novel RNA motif mediates the strict nuclear localization of a long noncoding RNA.

The ubiquitous presence of long noncoding RNAs (lncRNAs) in eukaryotes points to the importance of understanding how their sequences impact function. As many lncRNAs regulate nuclear events and thus must localize to nuclei, we analyzed the sequence requirements for nuclear localization in an intergenic lncRNA named BORG (BMP2-OP1-responsive gene), which is both spliced and polyadenylated but is strictly localized in nuclei. Subcellular localization of BORG was not dependent on the context or level of its expression or decay but rather depended on the sequence of the mature, spliced transcript. Mutational analyses indicated that nuclear localization of BORG was mediated through a novel RNA motif consisting of the pentamer sequence AGCCC with sequence restrictions at positions -8 (T or A) and -3 (G or C) relative to the first nucleotide of the pentamer. Mutation of the motif to a scrambled sequence resulted in complete loss of nuclear localization, while addition of even a single copy of the motif to a cytoplasmically localized RNA was sufficient to impart nuclear localization. Further, the presence of this motif in other cellular RNAs showed a direct correlation with nuclear localization, suggesting that the motif may act as a general nuclear localization signal for cellular RNAs.

Computational analysis of functional long noncoding RNAs reveals lack of peptide-coding capacity and parallels with 3' UTRs.

Recent transcriptome analyses have indicated that a large part of mammalian genomes are transcribed into long non-protein-coding RNAs (lncRNAs). However, only a very small fraction of them have been individually studied, and whether the majority of lncRNAs found in large-scale studies have a cellular role is debated. To gain insight into the sequence features and genomic architecture of the subset of lncRNAs that have been proven to be functional, we created a database containing studied lncRNAs manually culled from the literature along with a parallel database containing all annotated protein-coding human RNAs. The Functional lncRNA Database, which contains 204 lncRNAs and their splicing variants, is available at valadkhanlab.org/database. Analysis of the lncRNAs and their comparison to protein-coding transcripts revealed sequence features including paucity of introns and low GC content in lncRNAs, which could explain several biological characteristics of these transcripts, such as their nuclear localization and low expression level. The predicted ORFs in lncRNAs have poor start codon and ORF contexts, which would lead to activation of the nonsense-mediated decay pathways and thus make it unlikely for most lncRNAs to code for even short peptides. Interestingly, our analyses revealed significant similarities between the lncRNAs and the 3' untranslated regions (3' UTRs) in protein-coding RNAs in structural features and sequence composition. The presence of these intriguing parallels between the lncRNAs and 3' UTRs, which constitute the two main components of the RNA-mediated cellular regulatory system, indicates that highly similar evolutionary constraints govern the function of regulatory RNA sequences in the cell.

Effects of the fruit essential oil of Cuminum cyminum Linn. (Apiaceae) on pentylenetetrazol-induced epileptiform activity in F1 neurones of Helix aspersa.

The effect of the fruit essential oil of Cuminum cyminum Linn. (Apiaceae) (syn. Cuminum odorum Salisb) on the epileptiform activity induced by pentylenetetrazol (PTZ) was evaluated, using intracellular technique. The results demonstrated that extracellular application of the essential oil of Cuminum cyminum (1% and 3%) dramatically decreased the frequency of spontaneous activity induced by PTZ in a time and concentration dependent manner. In addition it showed protection against pentylenetetrazol-induced epileptic activity by increasing the duration, decreasing the amplitude of after hyperpolarization potential (AHP) following the action potential, the peak of action potential, and inhibition of the firing rate. These membrane effects suggest cellular mechanisms by which the essential oil of Cuminum cyminum can inhibit the PTZ-induced epileptic activity.