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Purpose To evaluate the imaging features of coronary spasm, including transluminal attenuation gradient (TAG) on coronary computed tomography angiography (CCTA), in patients with vasospastic angina (VA). Methods A total of 43 patients with a high clinical likelihood of VA were included in the study. All the subjects underwent double CCTA acquisition: CCTA without a vasodilator (‘baseline CT’) and CCTA during continuous intravenous nitrate infusion (‘IV nitrate CT’). A catheterized ergonovine provocation test was used to determine true VA patients. Coronary spasm is classified into focal- and diffuse-types according to morphological differences. We measured TAG and contrast enhancement of the proximal ostium (ProxHU) of each coronary artery for both the baseline and IV nitrate CT. Results Twenty-four patients (55.8%) showed positive results of coronary vasospasm on the provocation test. Thirty-eight vessels showed coronary spasms (29.5%): Focal-type in nine vessels (24%), and diffuse-type in 29 (76%). In the baseline CT, LCX showed significantly lower (steeper) TAG in spasm(+) vessels than in spasm(-) vessels, while LAD and RCA showed no significant differences in TAG. The ProxHU of LAD showed significantly lower values in spasm(+) vessels than in spasm(-) vessels, while the other vessels did not show significant differences in ProxHU. For IV nitrate CT, there were no significant differences in either the TAG and ProxHU between spasm(+) and (-) vessels for all the three vessel types. In subgroup analysis for spasm(+) vessels, diffuse spasms showed significantly lower TAG than focal spasms, while the ProxHU did not differ between the two types of spasm. Conclusions A relatively large percentage of coronary spasms present as diffuse type, and the TAG values significantly differed according to the morphological type of the coronary spasm.

Olfactory dysfunction and tinnitus are age-related otorhinolaryngological disorders with a high prevalence in the elderly population and share several common clinical features. However, there is no study investigating the relationship between these two diseases. We studied the prevalence of olfactory dysfunction and tinnitus among Koreans and studied the relationship between these two diseases based on the Korean National Health and Nutrition Examination Survey. The subjects of this study were enrolled from the Fifth Korean National Health and Nutrition Examination Survey (2010–2012, n = 25,534). Data of subjects aged 40 years and older who underwent physical examination and completed a self-reported questionnaire and other anthropometric variables were statistically analyzed. Odds ratios were calculated to identify the relationship between olfactory dysfunction and tinnitus, using multiple logistic regression models. Older males, non-smokers, non/lower alcohol drinker groups exhibited the relationship between olfactory dysfunction and tinnitus. Metabolic syndrome and mental health problems were associated with both olfactory dysfunction and tinnitus. After adjusting for confounding factors, olfactory dysfunction was significantly associated with tinnitus (OR 1.318). There was a dose-response relationship between tinnitus severity and the odds of olfactory dysfunction (ORs for mild, moderate and severe tinnitus were, respectively, 1.134, 1.569 and 2.044). Additional molecular genetics and animal studies are needed to determine the shared pathophysiology of the two diseases.

Advances in genomic technologies and the development of targeted therapeutics are making the use of precision medicine increasingly possible. In this study, we explored whether precision medicine can be applied for the management of refractory/relapsed pediatric solid tumors by discovering actionable alterations using targeted panel sequencing. Samples of refractory/relapsed pediatric solid tumors were tested using a targeted sequencing panel covering the exonic DNA sequences of 381 cancer genes and introns across 22 genes to detect clinically significant genomic aberrations in tumors. The molecular targets were tiered from 1 to 5 based on the presence of actionable genetic alterations, strength of supporting evidence, and drug availability in the Republic of Korea. From January 2016 to October 2018, 55 patients were enrolled. The median time from tissue acquisition to drug selection was 29 d (range 14-39), and tumor profiling was successful in 53 (96.4%) patients. A total of 27 actionable alterations in tiers 1-4 were detected in 20 patients (36.4%), and the majority of actionable alterations were copy number variations. The tiers of molecular alterations were tier 1 (clinical evidence) in 4 variants, tier 2 (preclinical evidence) in 8 variants, tier 3 (consensus opinion) in 2 variants, and tier 4 (actionable variants with a drug that is available in other countries but not in the Republic of Korea) in 9 variants. In one patient with relapsed neuroblastoma with ALK F1174L mutation and ALK amplification, lorlatinib was used in a compassionate use program, and it showed some efficacy. In conclusion, using a targeted sequencing panel to discover actionable alterations in relapsed/refractory pediatric solid tumors was practical and feasible.

Intratumoral heterogeneity (ITH) refers to the presence of distinct tumor cell populations. It provides vital information for the clinical prognosis, drug responsiveness, and personalized treatment of cancer patients. As genomic ITH in various cancers affects the expression patterns of genes, the expression profile could be utilized for determining ITH level. Herein, we present a novel approach to directly detect high ITH defined as a larger number of subclones from the gene expression pattern through machine learning approaches. We examined associations between gene expression profile and ITH of 12 cancer types from The Cancer Genome Atlas (TCGA) database. Using stomach adenocarcinoma (STAD) showing high association, we evaluated the performance of our method in predicting ITH by employing three machine learning algorithms using gene expression profile data. We classified tumors into high and low heterogeneity groups using the learning model through the selection of LASSO feature. The result showed that support vector machines (SVMs) outperformed other algorithms (AUC = 0.84 in SVMs and 0.82 in Naïve Bayes) and we were able to improve predictive power by using both combined data from mutation and expression. Furthermore, we evaluated the prediction ability of each model using simulation data generated by mixing cell lines of the Cancer Cell Line Encyclopedia (CCLE), and obtained consistent results with using real dataset. Our approach could be utilized for discriminating tumors with heterogeneous cell populations to characterize ITH.

Alternative polyadenylation (APA) in 3' untranslated regions (3' UTR) plays an important role in regulating transcript abundance, localization, and interaction with microRNAs. Length-variation of 3'UTRs by APA contributes to efficient proliferation of cancer cells. In this study, we investigated APA in single cancer cells and tumor microenvironment cells to understand the physiological implication of APA in different cell types. We analyzed APA patterns and the expression level of genes from the 515 single-cell RNA sequencing (scRNA-seq) dataset from 11 breast cancer patients. Although the overall 3'UTR length of individual genes was distributed equally in tumor and non-tumor cells, we found a differential pattern of polyadenylation in gene sets between tumor and non-tumor cells. In addition, we found a differential pattern of APA across tumor types using scRNA-seq data from 3 glioblastoma patients and 1 renal cell carcinoma patients. In detail, 1,176 gene sets and 53 genes showed the distinct pattern of 3'UTR shortening and over-expression as signatures for five cell types including B lymphocytes, T lymphocytes, myeloid cells, stromal cells, and breast cancer cells. Functional categories of gene sets for cellular proliferation demonstrated concordant regulation of APA and gene expression specific to cell types. The expression of APA genes in breast cancer was significantly correlated with the clinical outcome of earlier stage breast cancer patients. We identified cell type-specific APA in single cells, which allows the identification of cell types based on 3'UTR length variation in combination with gene expression. Specifically, an immune-specific APA signature in breast cancer could be utilized as a prognostic marker of early stage breast cancer.

CDH23 mutations have mostly been associated with prelingual severe-to-profound sensorineural hearing loss (SNHL) in either syndromic or nonsyndromic SNHL (DFNB12). Herein, we demonstrate the contribution of CDH23 mutations to postlingual nonsyndromic SNHL (NS-SNHL). We screened 32 Korean adult probands with postlingual NS-SNHL sporadically or in autosomal recessive fashion using targeted panel or whole exome sequencing. We identified four (12.5%, 4/32) potential postlingual DFNB12 families that segregated the recessive CDH23 variants, qualifying for our criteria along with rapidly progressive SNHL. Three of the four families carried one definite pathogenic CDH23 variant previously known as the prelingual DFNB12 variant in a trans configuration with rare CDH23 variants. To determine the contribution of rare CDH23 variants to the postlingual NS-SNHL, we checked the minor allele frequency (MAF) of CDH23 variants detected from our postlingual NS-SNHL cohort and prelingual NS-SNHL cohort, among the 2040 normal control chromosomes. The allele frequency of these CDH23 variants in our postlingual cohort was 12.5%, which was significantly higher than that of the 2040 control chromosomes (5.53%), confirming the contribution of these rare CDH23 variants to postlingual NS-SNHL. Furthermore, MAF of rare CDH23 variants from the postlingual NS-SNHL group was significantly higher than that from the prelingual NS-SNHL group. This study demonstrates an important contribution of CDH23 mutations to poslingual NS-SNHL and shows that the phenotypic spectrum of DFNB12 can be broadened even into the presbycusis, depending on the pathogenic potential of variants. We also propose that pathogenic potential of CDH23 variants and the clinical fate of DFNB12 may be predicted by MAF.

Epithelial-mesenchymal transition (EMT) is an important parameter related to breast cancer survival. Among several microRNAs predicted to target EMT-related genes, miR-506 is a novel miRNA found to be significantly related to breast cancer patient survival in a meta-analysis. miR-506 suppressed the expression of mesenchymal genes such as Vimentin, Snai2, and CD151 in MDA-MB-231 human breast cancer cell line. Moreover, NF-kappa B bound to the upstream promoter region of miR-506 to suppress transcription. Overexpression of miR-506 inhibited TGF beta-induced EMT and suppressed adhesion, invasion, and migration of MDA-MB-231 cells. From these results, we concluded that miR-506 plays a key role in the process of EMT through posttranslational control of EMT-related genes.

A single tumor biopsy specimen is typically used in cancer genome studies. However, it may represent incompletely the underlying mutational and transcriptional profiles of tumor biology. Multi-regional biopsies have the advantage of increased sensitivity for genomic profiling, but they are not cost-effective. The concept of an alternative method such as the pooling of multiple biopsies is a challenge. In order to determine if the pooling of distinct regions is representative at the genomic and transcriptome level, we performed sequencing of four regional samples and pooled samples for four cancer types including colon, stomach, kidney and liver cancer. Subsequently, a comparative analysis was conducted to explore differences in mutations and gene expression profiles between multiple regional biopsies and pooled biopsy for each tumor. Our analysis revealed a marginal level of regional difference in detected variants, but in those with low allele frequency, considerable discrepancies were observed. In conclusion, sequencing pooled samples has the benefit of detecting many variants with moderate allele frequency that occur in partial regions, but it is not applicable for detecting low-frequency mutations that require deep sequencing.

Radiofrequency (RF) radiation does not transfer high energy to break the covalent bonds of macromolecules, but these low energy stimuli might be sufficient to induce molecular responses in a specific manner. We monitored the effect of 1,763 MHz RF radiation on cultured human dermal papilla cells (hDPCs) by evaluating changes in the expression of cytokines related to hair growth. The expression of insulin-like growth factor-1 (IGF-1) mRNA in hDPCs was significantly induced upon RF radiation at the specific absorption rate of 10 W/kg, which resulted in increased expression of B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL-2) and cyclin D1 (CCND1) proteins and increased phosphorylation of MAPK1 protein. Exposure to 10 W/kg RF radiation 1 h per day for 7 days significantly enhanced hair shaft elongation in ex vivo hair organ cultures. In RF-exposed follicular matrix keratinocytes in the hair bulb, the expression of Ki-67 was increased, while the signal for terminal deoxynucleotidyl transferase dUTP nick end labeling was reduced. From these results, we suggest that 1,763 MHz RF exposure stimulates hair growth in vitro through the induction of IGF-1 in hDPCs.

MicroRNA regulates cellular responses to ionizing radiation (IR) through translational control of target genes. We analyzed time-series changes in microRNA expression following γ-irradiation in H1299 lung cancer cells using microarray analysis. Significantly changed IR-responsive microRNAs were selected based on analysis of variance analysis, and predicted target mRNAs were enriched in mitogen-activated protein kinase (MAPK) signaling. Concurrent analysis of time-series mRNA and microRNA profiles uncovered that expression of miR-26b was down regulated, and its target activating transcription factor 2 (ATF2) mRNA was up regulated in γ-irradiated H1299 cells. IR in miR-26b overexpressed H1299 cells could not induce expression of ATF2. When c-Jun N-terminal kinase activity was inhibited using SP600125, expression of miR-26b was induced following γ-irradiation in H1299 cells. From these results, we concluded that IR-induced up-regulation of ATF2 was coordinately enhanced by suppression of miR-26b in lung cancer cells, which may enhance the effect of IR in the MAPK signaling pathway.

Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs) using a machine learning method inspired by the “Divide & Conquer” strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target genes and/or the promoter architectures resulting from the interaction of those binding sites with the RNA polymerase., The diversity of life forms frequently results from small changes in the regulatory systems that control gene expression. These changes often occur in cis-elements relevant to transcriptional regulation that are difficult to discern, as they are short, and are embedded in a genomic background that does not play a direct role in gene expression, or that consists of disparate sequences such as those from horizontally acquired genes. We devised a machine-learning method that significantly improves the identification of these elements, uncovering families of binding site motifs (i.e., “submotifs”), instead of a single consensus recognized by a transcriptional regulator. The method can also incorporate other cis-elements to fully describe promoter architectures. Far from being just a computational convenience, ChIP-chip and custom expression microarray experiments for the PhoP regulon validated the high conservation and modular evolution of submotifs throughout the gamma/enterobacteria. This suggests that the major cause of divergence between species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous and horizontally-acquired target genes, and/or to the uncovered promoter architectures governing the interaction between the regulator and the RNA polymerase., This work was supported in part by the Spanish Ministry of Science and Technology under project TIN2006-12879 and TIN2009-13950 by Consejeria de Innovacion, Investigacion y Ciencia de la de la Junta de Andalucia under project TIC02788. E.A.G. is an Investigator of the Howard Hughes Medical Institute.

The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5’ fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy., Author Summary Little is known about the molecular biology of medullary thyroid cancer (MTC), which is a rare disease. Genomics are increasingly being used to improve our knowledge about disease biology and to identify therapeutic targets in many cancers. Here, we report the largest genomic results of MTC to date. MTC tissue frequently included several mutations. For the first time, anaplastic lymphoma kinase (ALK) rearrangements were detected in MTC: one case with a glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK fusion, and another case with an echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The fusion mechanism of the novel GFPT1-ALK fusion was successfully investigated using molecular biology techniques. In addition, an inhibitor of ALK (crizotinib) dramatically decreased the number of metastatic MTC lesions harboring the EML4-ALK fusion, thus verifying the fusion as a promising target in MTC. Our findings suggest that using rapidly improving sequencing techniques and accumulated genomic data to comprehensively perform genetic analyses on rare tumors, such as MTC, will help to improve the poor prognosis of orphan diseases.

The immunoglobulin (Ig)-like domain containing receptor 1 (ILDR1) gene encodes angulin-2/ILDR1, a recently discovered tight junction protein, which forms tricellular tight junction (tTJ) structures with tricellulin and lipolysis-stimulated lipoprotein receptor (LSR) at tricellular contacts (TCs) in the inner ear. Previously reported recessive mutations within ILDR1 have been shown to cause severe to profound nonsyndromic sensorineural hearing loss (SNHL), DFNB42. Whole-exome sequencing of a Korean multiplex family segregating partial deafness identified a novel homozygous ILDR1 variant (p.P69H) within the Ig-like domain. To address the pathogenicity of p. P69H, the angulin-2/ILDR1 p.P69H variant protein, along with the previously reported pathogenic ILDR1 mutations, was expressed in angulin-1/LSR knockdown epithelial cells. Interestingly, partial mislocalization of the p.P69H variant protein and tricellulin at TCs was observed, in contrast to a severe mislocalization and complete failure of tricellulin recruitment of the other reported ILDR1 mutations. Additionally, three-dimensional protein modeling revealed that angulin-2/ILDR1 contributed to tTJ by forming a homo-trimer structure through its Ig-like domain, and the p.P69H variant was predicted to disturb homo-trimer formation. In this study, we propose a possible role of angulin-2/ILDR1 in tTJ formation in the inner ear and a wider audiologic phenotypic spectrum of DFNB42 caused by mutations within ILDR1.