GenoMycAnalyzer: a web-based tool for species and drug resistance prediction for Mycobacterium genomes.

BACKGROUND: Drug-resistant tuberculosis (TB) is a major threat to global public health. Whole-genome sequencing (WGS) is a useful tool for species identification and drug resistance prediction, and many clinical laboratories are transitioning to WGS as a routine diagnostic tool. However, user-friendly and high-confidence automated bioinformatics tools are needed to rapidly identify M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), detect drug resistance, and further guide treatment options. RESULTS: We developed GenoMycAnalyzer, a web-based software that integrates functions for identifying MTBC and NTM species, lineage and spoligotype prediction, variant calling, annotation, drug-resistance determination, and data visualization. The accuracy of GenoMycAnalyzer for genotypic drug susceptibility testing (gDST) was evaluated using 5,473 MTBC isolates that underwent phenotypic DST (pDST). The GenoMycAnalyzer database was built to predict the gDST for 15 antituberculosis drugs using the World Health Organization mutational catalogue. Compared to pDST, the sensitivity of drug susceptibilities by the GenoMycAnalyzer for first-line drugs ranged from 95.9% for rifampicin (95% CI 94.8-96.7%) to 79.6% for pyrazinamide (95% CI 76.9-82.2%), whereas those for second-line drugs ranged from 98.2% for levofloxacin (95% CI 90.1-100.0%) to 74.9% for capreomycin (95% CI 69.3-80.0%). Notably, the integration of large deletions of the four resistance-conferring genes increased gDST sensitivity. The specificity of drug susceptibilities by the GenoMycAnalyzer ranged from 98.7% for amikacin (95% CI 97.8-99.3%) to 79.5% for ethionamide (95% CI 76.4-82.3%). The incorporated Kraken2 software identified 1,284 mycobacterial species with an accuracy of 98.8%. GenoMycAnalyzer also perfectly predicted lineages for 1,935 MTBC and spoligotypes for 54 MTBC. CONCLUSIONS: GenoMycAnalyzer offers both web-based and graphical user interfaces, which can help biologists with limited access to high-performance computing systems or limited bioinformatics skills. By streamlining the interpretation of WGS data, the GenoMycAnalyzer has the potential to significantly impact TB management and contribute to global efforts to combat this infectious disease. GenoMycAnalyzer is available at http://www.mycochase.org .

Establishment of tumor microenvironment-preserving organoid model from patients with intracranial meningioma.

BACKGROUND: Although meningioma is the most common primary brain tumor, treatments rely on surgery and radiotherapy, and recurrent meningiomas have no standard therapeutic options due to a lack of clinically relevant research models. Current meningioma cell lines or organoids cannot reflect biological features of patient tumors since they undergo transformation along culture and consist of only tumor cells without microenvironment. We aim to establish patient-derived meningioma organoids (MNOs) preserving diverse cell types representative of the tumor microenvironment. METHODS: The biological features of MNOs were evaluated using WST, LDH, and collagen-based 3D invasion assays. Cellular identities in MNOs were confirmed by immunohistochemistry (IHC). Genetic alteration profiles of MNOs and their corresponding parental tumors were obtained by whole-exome sequencing. RESULTS: MNOs were established from four patients with meningioma (two grade 1 and two grade 2) at a 100% succession rate. Exclusion of enzymatic dissociation-reaggregation steps endowed MNOs with original histology and tumor microenvironment. In addition, we used a liquid media culture system instead of embedding samples into Matrigel, resulting in an easy-to-handle, cost-efficient, and time-saving system. MNOs maintained their functionality and morphology after long-term culture (> 9 wk) and repeated cryopreserving-recovery cycles. The similarities between MNOs and their corresponding parental tumors were confirmed by both IHC and whole-exome sequencing. As a representative application, we utilized MNOs in drug screening, and mifepristone, an antagonist of progesterone receptor, showed prominent antitumor efficacy with respect to viability, invasiveness, and protein expression. CONCLUSION: Taken together, our MNO model overcame limitations of previous meningioma models and showed superior resemblance to parental tumors. Thus, our model could facilitate translational research identifying and selecting drugs for meningioma in the era of precision medicine.

Single-cell profiling identifies distinct hormonal, immunologic, and inflammatory signatures of endometriosis-constituting cells.

Endometriosis consists of ectopic endometrial epithelial cells (EEECs) and ectopic endometrial stromal cells (EESCs) mixed with heterogeneous stromal cells. To address how endometriosis-constituting cells are different from normal endometrium and among endometriosis subtypes and how their molecular signatures are related to phenotypic manifestations, we analyzed ovarian endometrial cyst (OEC), superficial peritoneal endometriosis (SPE), and deep infiltrating endometriosis (DIE) from 12 patients using single-cell RNA-sequencing (scRNA-seq). We identified 11 cell clusters, including EEEC, EESC, fibroblasts, inflammatory/immune, endothelial, mesothelial, and Schwann cells. For hormonal signatures, EESCs, but not EEECs, showed high estrogen signatures (estrogen response scores and HOXA downregulation) and low progesterone signatures (DKK1 downregulation) compared to normal endometrium. In EEECs, we found MUC5B+ TFF3low cells enriched in endometriosis. In lymphoid cells, evidence for both immune activation (high cytotoxicity in NK) and exhaustion (high checkpoint genes in NKT and cytotoxic T) was identified in endometriosis. Signatures and subpopulations of macrophages were remarkably different among endometriosis subtypes with increased monocyte-derived macrophages and IL1B expression in DIE. The scRNA-seq predicted NRG1 (macrophage)-ERBB3 (Schwann cell) interaction in endometriosis, expressions of which were validated by immunohistochemistry. Myofibroblast subpopulations differed according to the location (OECs from fibroblasts and SPE/DIEs from mesothelial cells and fibroblasts). Endometriosis endothelial cells displayed proinflammation, angiogenesis, and leaky permeability signatures that were enhanced in DIE. Collectively, our study revealed that (1) many cell types-endometrial, lymphoid, macrophage, fibroblast, and endothelial cells-are altered in endometriosis; (2) endometriosis cells show estrogen responsiveness, immunologic cytotoxicity and exhaustion, and proinflammation signatures that are different in endometriosis subtypes; and (3) novel endometriosis-specific findings of MUC5B+ EEECs, mesothelial cell-derived myofibroblasts, and NRG1-ERBB3 interaction may underlie the pathogenesis of endometriosis. Our results may help extend pathologic insights, dissect aggressive diseases, and discover therapeutic targets in endometriosis. © 2023 The Pathological Society of Great Britain and Ireland.

Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism.

Substantial evidence suggests that chromosomal abnormalities contribute to the risk of autism. The duplication of human chromosome 15q11-13 is known to be the most frequent cytogenetic abnormality in autism. We have modeled this genetic change in mice by using chromosome engineering to generate a 6.3 Mb duplication of the conserved linkage group on mouse chromosome 7. Mice with a paternal duplication display poor social interaction, behavioral inflexibility, abnormal ultrasonic vocalizations, and correlates of anxiety. An increased MBII52 snoRNA within the duplicated region, affecting the serotonin 2c receptor (5-HT2cR), correlates with altered intracellular Ca(2+) responses elicited by a 5-HT2cR agonist in neurons of mice with a paternal duplication. This chromosome-engineered mouse model for autism seems to replicate various aspects of human autistic phenotypes and validates the relevance of the human chromosome abnormality. This model will facilitate forward genetics of developmental brain disorders and serve as an invaluable tool for therapeutic development.

Integrated Bioinformatics Analysis Identified ASNS and DDIT3 as the Therapeutic Target in Castrate-Resistant Prostate Cancer.

Many studies have demonstrated the mechanisms of progression to castration-resistant prostate cancer (CRPC) and novel strategies for its treatment. Despite these advances, the molecular mechanisms underlying the progression to CRPC remain unclear, and currently, no effective treatments for CRPC are available. Here, we characterized the key genes involved in CRPC progression to gain insight into potential therapeutic targets. Bicalutamide-resistant prostate cancer cells derived from LNCaP were generated and named Bical R. RNA sequencing was used to identify differentially expressed genes (DEGs) between LNCaP and Bical R. In total, 631 DEGs (302 upregulated genes and 329 downregulated genes) were identified. The Cytohubba plug-in in Cytoscape was used to identify seven hub genes (ASNS, AGT, ATF3, ATF4, DDIT3, EFNA5, and VEGFA) associated with CRPC progression. Among these hub genes, ASNS and DDIT3 were markedly upregulated in CRPC cell lines and CRPC patient samples. The patients with high expression of ASNS and DDIT3 showed worse disease-free survival in patients with The Cancer Genome Atlas (TCGA)-prostate adenocarcinoma (PRAD) datasets. Our study revealed a potential association between ASNS and DDIT3 and the progression to CRPC. These results may contribute to the development of potential therapeutic targets and mechanisms underlying CRPC progression, aiming to improve clinical efficacy in CRPC treatment.

Single-cell RNA sequencing of a poorly metastatic melanoma cell line and its subclones with high lung and brain metastasis potential reveals gene expression signature of metastasis with prognostic implication.

The molecular mechanisms underlying melanoma metastasis remain poorly understood. In this study, we aimed to delineate the mechanisms underlying gene expression alterations during metastatic potential acquisition and characterize the metastatic subclones within primary cell lines. We performed single-cell RNA sequencing of a poorly metastatic melanoma cell line (WM239A) and its subclones with high metastatic potential to the lung (113/6-4L) and the brain (131/4-5B1 and 131/4-5B2). Unsupervised clustering of 8173 melanoma cells identified three distinct clusters according to cell type ('Primary', 'Lung' and 'Brain' clusters) with differential expression of MITF and AXL pathways and putative cancer and cell cycle drivers, with the lung cluster expressing intermediate but distinct gene profiles between primary and brain clusters. Principal component (PC) analysis revealed that PC2 (the second PC), which was positively associated with MITF expression and negatively with AXL pathways, primarily segregated cell types, in addition to PC1 of the cell cycle pathway. Pseudotime trajectory and RNA velocity analyses suggested the existence of cellular subsets with metastatic potential in the Primary cluster and an association between PC2 signature alteration and metastasis potential acquisition. Analysis of The Cancer Genome Atlas melanoma samples by clustering into PC2-high and -low clusters by quartiles of PC2 signature expression revealed that the PC2-high cluster was an independent significant factor for poor prognosis (p-value = 0.003) with distinct genomic and transcriptomic characteristics, compared to the PC2-low cluster. In conclusion, we identified signatures of melanoma metastasis with prognostic significance and putative pro-metastatic subclones within a primary cell line.

Targeted deep sequencing reveals genomic alterations of actinic keratosis/cutaneous squamous cell carcinoma in situ and cutaneous squamous cell carcinoma.

Actinic keratosis (AK) and cutaneous squamous cell carcinoma in situ (CIS) are two of the most common precursors of cutaneous squamous cell carcinoma (cSCC). However, the genomic landscape of AK/CIS and the drivers of cSCC progression remain to be elucidated. The aim of our study was to investigate the genomic alterations between AK/CIS and cSCC in terms of somatic mutations and copy number alterations (CNAs). We performed targeted deep sequencing of 160 cancer-related genes with a median coverage of 515× for AK (N = 9), CIS (N = 9), cSCC lesions (N = 13), and matched germline controls from 17 patients. cSCC harboured higher abundance of total mutations, driver mutations and CNAs than AK/CIS. Driver mutations were found in TP53 (81%), NOTCH1 (32%), RB1 (26%) and CDKN2A (19%). All AK/CIS and cSCC lesions (93.5%), except two, harboured TP53 or NOTCH1 mutations, some of which were known oncogenic mutations or reported mutations in normal skin. RB1 driver mutations were found in CIS/cSCC (36.4%) but not in AK. CDKN2A driver mutations were found more frequently in cSCC (30.8%) than in AK/CIS (11.1%). Among recurrent (≥3 samples) CNAs (gain in MYC and PIK3CA/SOX2/TP63; loss in CDKN2A and RB1), MYC (8q) gain and CDKN2A (9p) loss were more frequently detected in cSCC (30.8%) than in AK/CIS (11.1%). Ultraviolet was responsible for the majority of somatic mutations in both AK/CIS and cSCC. Our study revealed that AK/CIS lesions harbour prevalent TP53 or NOTCH1 mutations and that additional somatic mutations and CNAs may lead to cSCC progression in AK/CIS lesions.

Molecular genetic evidence supporting diverse histogenic origins of germ cell tumors.

Germ cell tumors (GCTs) originate during the histogenesis of primordial germ cells to mature gametes. Previous studies identified five histogenic mechanisms in ovarian mature teratomas (type I: failure of meiosis I; type II: failure of meiosis II; type III: duplication of the genome of a mature gamete; type IV: no meiosis; and type V: fusion of two different ova), but those of other GCTs remain elusive. In this study, we analyzed 84 GCTs of various pathologic types to identify the histogenesis using single-nucleotide polymorphism array by analyzing copy-neutral loss of heterozygosity (CN-LOH) and copy number alterations (CNAs). We detected types I and II in ovarian teratomas, type III in ovarian teratomas and yolk sac tumors (YSTs), and type IV in all GCT types. The GCTs with multiple-type histogenesis (I-IV) (ovarian mature/immature teratomas and YST) show meiotic CN-LOH with scant CNAs. Type IV-only GCTs are either with mitotic CN-LOH and abundant CNAs (seminoma, dysgerminoma, testicular mixed GCTs) or with scant CNAs and no CN-LOH (pediatric testicular and mediastinal teratomas). The development sequences of CN-LOH and CNA are different between the multiple type (I-IV) GCTs and type IV-only GCTs. We analyzed two different histologic areas in eight GCTs (one mature teratoma with a mucin-secreting adenoma, two immature teratomas, and five mixed GCTs). We found that GCTs (mature teratoma, immature teratoma, and mixed GCT) showed different genomic alterations between histologic areas, suggesting that genomic differences within a GCT could accompany histologic differentiation. Of note, we found evidence for collision tumors in a mixed GCT. Our data indicate that GCTs may have various histogenesis and intratumoral genomic differences, which might provide important information for the identification of GCTs, especially for those with different histologic areas. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

tReasure: R-based GUI package analyzing tRNA expression profiles from small RNA sequencing data.

BACKGROUND: Recent deep sequencing technologies have proven to be valuable resources to gain insights into the expression profiles of diverse tRNAs. However, despite these technologies, the association of tRNAs with diverse diseases has not been explored in depth because analytical tools are lacking. RESULTS: We developed a user-friendly tool, tRNA Expression Analysis Software Utilizing R for Easy use (tReasure), to analyze differentially expressed tRNAs (DEtRNAs) from deep sequencing data of small RNAs using R packages. tReasure can quantify individual mature tRNAs, isodecoders, and isoacceptors. By adopting stringent mapping strategies, tReasure supports the precise measurement of mature tRNA read counts. The whole analysis workflow for determining DEtRNAs (uploading FASTQ files, removing adapter sequences and poor-quality reads, mapping and quantifying tRNAs, filtering out low count tRNAs, determining DEtRNAs, and visualizing statistical analysis) can be performed with the tReasure package. CONCLUSIONS: tReasure is an open-source software available for download at https://treasure.pmrc.re.kr and will be indispensable for users who have little experience with command-line software to explore the biological implication of tRNA expression.

Single-cell RNA sequencing reveals the existence of pro-metastatic subpopulation within a parental B16 murine melanoma cell line.

The mechanism of melanoma metastasis is poorly understood, especially at the single-cell level. To understand the evolution from primary melanoma to metastasis, we investigated single-cell transcriptome profiles of parental B16 melanoma cells (B16F0) and its highly metastatic subclone (B16F10). Genomic alterations between cells were also analyzed by whole-exome sequencing. We identified 274 differentially expressed genes (DEGs) in B16F10, including upregulated genes related to metastasis, Lgals3, Sparc, Met, and Tmsb4x, and downregulated Mitf pathway genes, Ptgds, Cyb5a, and Cd63. The proportion of cycling cells and cells highly expressing Kdm5b was significantly high in B16F10 cells. Among the five subclusters of B16 cells (C1-5), C3/C4 clusters comprised both B16F0 and B16F10 cells and exhibited intermediate DEG patterns, whereas the C5 cluster mostly comprised B16F10 and showed typical metastatic characteristics. In trajectory analysis, the C4 cluster in B16F0, which showed unique characteristics (mainly cycling cells and upregulation of Mitf pathway genes), have transition potential to the C5 cluster (B16F10). Regarding genomic alterations, stepwise evolution with shared mutations, including Braf, Pten, and Trp53, and further specific alterations led to metastatic development. Our results provide deeper understanding of melanoma metastasis at the single-cell level, thus aiding further studies in melanoma metastasis control.

TNNC1 knockout reverses metastatic potential of ovarian cancer cells by inactivating epithelial-mesenchymal transition and suppressing F-actin polymerization.

Troponin C type 1 (TNNC1) is commonly overexpressed in ovarian cancer. However, the biological implications of TNNC1 overexpression on ovarian cancer malignization and its related mechanism remain unknown. To elucidate these implications, we knocked out the TNNC1 gene in TNNC1-overexpressing SKOV-3-13 ovarian cancer cells using CRISPR/Cas-9 technology and observed the changes in metastatic phenotypes and related molecular pathways. TNNC1-knockout (KO) cells showed significantly reduced proliferation and colony formation when compared with TNNC1 wild-type cells (P < 0.01). In TNNC1-KO cells, wound healing, migration, and invasive phenotypes decreased. Upon observation of upstream regulators of epithelial-mesenchymal transition (EMT), levels of phosphorylated AKT (Ser-473 and Thr-308) and GSK-3ß (inactive form) were found to be decreased in TNNC1-KO cells. Accordingly, SNAIL and SLUG expression decreased and were almost completely localized in the cytoplasm following TNNC1 silencing. Regarding downstream EMT markers, N-cadherin and vimentin expression decreased, but E-cadherin expression increased. Related matrix metalloproteinase and chemokine expression generally decreased. TNNC1 deficiency also suppressed F-actin polymerization. In conclusion, TNNC1 overexpression contributes to the metastatic behavior of ovarian cancer by perturbation of EMT and actin microfilaments. Our results provide a better understanding of the detailed molecular mechanism of ovarian cancer metastasis associated with TNNC1 overexpression.

Pathogenic NF1 truncating mutation and copy number alterations in a dedifferentiated liposarcoma with multiple lung metastasis: a case report.

BACKGROUND: Dedifferentiated liposarcoma (DDLPS), which accounts for an estimated 15-20% of liposarcomas, is a high-grade and aggressive malignant neoplasm, exhibiting a poor response to available therapeutic agents. However, genetic alteration profiles of DDLPS as well as the role of NF1 mutations have not been studied extensively. CASE PRESENTATION: The current study reports a patient presenting with rapidly growing DDLPS accompanied by multiple lung and pleural metastases, in whom whole-exome sequencing revealed a NF1 truncating mutation of the known pathogenic variant, c.C7486T, p.R2496X, as well as multiple copy number alterations (CNAs), including the well-known 12q13-15 amplification, and multiple chromothripsis events encompassing potential cancer-related genes. CONCLUSIONS: Our results suggest that, in addition to the 12q13-15 amplification, NF1 inactivation mutation and other CNAs may contribute to DDLPS tumorigenesis accompanied by aggressive clinical features.

Co-introduction of plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, increases fitness and virulence of bacterial host.

BACKGROUND: Bacterial isolates with multiple plasmids harbouring different carbapenemase genes have emerged and been identified repeatedly, despite a general notion that plasmids confer fitness cost in bacterial host. In this study, we investigated the effects of plasmids with carbapenemase genes on the fitness and virulence of bacteria. METHODS: Different plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, were isolated from a carbapenem-resistant K. pneumoniae strain. Each plasmid was conjugated into the Escherichia coli strain DH5α, and a transconjugant with both plasmids was also obtained by transformation. Their in vitro competitive ability, biofilm formation, serum resistance, survival ability within macrophage and fruit fly, and fly killing ability were evaluated. RESULTS: The transconjugants with a single plasmid showed identical phenotypes to the plasmid-free strain, except that they decreased fly survival after infection. However, significantly increased fitness, virulence and biofilm production were observed consistently for the transconjugant with both plasmids, harbouring blaNDM-1 and blaOXA-232. CONCLUSIONS: Our data indicate that bacteria carrying multiple plasmids encoding different carbapenemases may have increased fitness and virulence, emphasizing the need for diverse strategies to combat antimicrobial resistance.

Evolution of Klebsiella pneumoniae with mucoid and non-mucoid type colonies within a single patient.

We obtained nine Klebsiella pneumoniae isolates successively isolated from a single patient. Four pairs (M1-M4 and NM1-NM4) obtained simultaneously from the same site showed different colony types, mucoid and non-mucoid, while the final isolate (M5) was isolated alone from the blood and showed a mucoid phenotype. The whole genome of isolate M5 was sequenced de novo using the PacBio RSII system, while the others were sequenced with an Illumina Hiseq4000 and mapped to the genome sequences of M5. To identify insertions or deletions in the cps locus, we amplified and sequenced cps locus genes. We identified insertion sequence (IS) elements in several genes of the cps locus or one amino acid substitution in WcaJ in all non-mucoid isolates. Five additional amino acid alterations in RpsJ, LolE, Lon-2, PpsE, and a hypothetical protein were detected in some mucoid and non-mucoid isolates. Based on the genome data and cps locus sequences, the mucoid phenotype may have been lost or converted into the non-mucoid phenotype because of the insertion of IS elements or amino acid alterations at this locus. We inferred a within-host evolutionary scenario, in which non-mucoid variants emerged repeatedly from mucoid isolates, but may be short-lived because of their low fitness.

Whole-exome sequencing identifies recurrent AKT1 mutations in sclerosing hemangioma of lung.

Pulmonary sclerosing hemangioma (PSH) is a benign tumor with two cell populations (epithelial and stromal cells), for which genomic profiles remain unknown. We conducted exome sequencing of 44 PSHs and identified recurrent somatic mutations of AKT1 (43.2%) and ß-catenin (4.5%). We used a second subset of 24 PSHs to confirm the high frequency of AKT1 mutations (overall 31/68, 45.6%; p.E17K, 33.8%) and recurrent ß-catenin mutations (overall 3 of 68, 4.4%). Of the PSHs without AKT1 mutations, two exhibited AKT1 copy gain. AKT1 mutations existed in both epithelial and stromal cells. In two separate PSHs from one patient, we observed two different AKT1 mutations, indicating they were not disseminated but independent arising tumors. Because the AKT1 mutations were not found to co-occur with ß-catenin mutations (or any other known driver alterations) in any of the PSHs studied, we speculate that this may be the single-most common driver alteration to develop PSHs. Our study revealed genomic differences between PSHs and lung adenocarcinomas, including a high rate of AKT1 mutation in PSHs. These genomic features of PSH identified in the present study provide clues to understanding the biology of PSH and for differential genomic diagnosis of lung tumors.

A 3.4-kb Copy-Number Deletion near EPAS1 Is Significantly Enriched in High-Altitude Tibetans but Absent from the Denisovan Sequence.

Tibetan high-altitude adaptation (HAA) has been studied extensively, and many candidate genes have been reported. Subsequent efforts targeting HAA functional variants, however, have not been that successful (e.g., no functional variant has been suggested for the top candidate HAA gene, EPAS1). With WinXPCNVer, a method developed in this study, we detected in microarray data a Tibetan-enriched deletion (TED) carried by 90% of Tibetans; 50% were homozygous for the deletion, whereas only 3% carried the TED and 0% carried the homozygous deletion in 2,792 worldwide samples (p < 10(-15)). We employed long PCR and Sanger sequencing technologies to determine the exact copy number and breakpoints of the TED in 70 additional Tibetan and 182 diverse samples. The TED had identical boundaries (chr2: 46,694,276-46,697,683; hg19) and was 80 kb downstream of EPAS1. Notably, the TED was in strong linkage disequilibrium (LD; r(2) = 0.8) with EPAS1 variants associated with reduced blood concentrations of hemoglobin. It was also in complete LD with the 5-SNP motif, which was suspected to be introgressed from Denisovans, but the deletion itself was absent from the Denisovan sequence. Correspondingly, we detected that footprints of positive selection for the TED occurred 12,803 (95% confidence interval = 12,075-14,725) years ago. We further whole-genome deep sequenced (>60×) seven Tibetans and verified the TED but failed to identify any other copy-number variations with comparable patterns, giving this TED top priority for further study. We speculate that the specific patterns of the TED resulted from its own functionality in HAA of Tibetans or LD with a functional variant of EPAS1.

A missense mutation in EBF2 was segregated with imperforate anus in a family across three generations.

The etiology of imperforate anus, a major phenotype of anorectal malformation (ARM), is still unknown and not a single gene has been reported to be associated with it. We studied a Korean family with six affected members with imperforate anus across three generations by whole exome sequencing and identified a missense mutation in the EBF2 gene (c.215C > T; p.Ala72Val). This mutation is completely segregated with the disease phenotype in the family and is evolutionarily highly conserved among diverse vertebrates. Also, this mutation was predicted to be functionally damaging. These results support that missense mutation in the EBF2 c.215C > T (p.Ala72Val) is very likely to contribute to the pathogenesis of ARM in this family.

Intraindividual genomic heterogeneity of high-grade serous carcinoma of the ovary and clinical utility of ascitic cancer cells for mutation profiling.

Intraindividual tumoural heterogeneity (ITH) is a hallmark of solid tumours and impedes accurate genomic diagnosis and selection of proper therapy. The aim of this study was to identify ITH of ovarian high-grade serous carcinomas (OSCs) and to determine the utility of ascitic cancer cells as a resource for mutation profiling in spite of ITH. We performed whole-exome sequencing, copy number profiling and DNA methylation profiling of four OSC genomes by using multiregional biopsies from 13 intraovarian lesions, 12 extraovarian tumour lesions (omentum/peritoneum), and ascitic cells. We observed substantial levels of heterogeneity in mutations and copy number alterations (CNAs) of the OSCs. We categorized the mutations into 'common', 'shared' and 'private' according to the regional distribution. Six common, eight shared and 24 private mutations were observed in known cancer-related genes. Common mutations had a higher mutant allele frequency, and included TP53 mutations in all four OSCs. Region-specific chromosomal amplifications and deletions involving BRCA1, PIK3CA and RB1 were also identified. It is of note that the mutations detected in ascitic cancer cells represented 92.3-100% of overall somatic mutations in the given case. Phylogenetic analyses of ascitic genomes predicted a polyseeding origin of somatic mutations in ascitic cells. Our results demonstrate that, despite ITH, somatic mutations, CNAs and DNA methylations in both 'common' category and cancer-related genes were highly conserved in ascitic cells of OSCs, highlighting the clinical relevance of genome analysis of ascitic cells. Ascitic tumour cells may serve as a potential resource for discovering somatic mutations of primary OSC with diagnostic and therapeutic relevance. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic structure, divergence and admixture of Han Chinese, Japanese and Korean populations.

BACKGROUND: Han Chinese, Japanese and Korean, the three major ethnic groups of East Asia, share many similarities in appearance, language and culture etc., but their genetic relationships, divergence times and subsequent genetic exchanges have not been well studied. RESULTS: We conducted a genome-wide study and evaluated the population structure of 182 Han Chinese, 90 Japanese and 100 Korean individuals, together with the data of 630 individuals representing 8 populations wordwide. Our analyses revealed that Han Chinese, Japanese and Korean populations have distinct genetic makeup and can be well distinguished based on either the genome wide data or a panel of ancestry informative markers (AIMs). Their genetic structure corresponds well to their geographical distributions, indicating geographical isolation played a critical role in driving population differentiation in East Asia. The most recent common ancestor of the three populations was dated back to 3000 ~ 3600 years ago. Our analyses also revealed substantial admixture within the three populations which occurred subsequent to initial splits, and distinct gene introgression from surrounding populations, of which northern ancestral component is dominant. CONCLUSIONS: These estimations and findings facilitate to understanding population history and mechanism of human genetic diversity in East Asia, and have implications for both evolutionary and medical studies.

Leukocyte-specific protein 1 regulates T-cell migration in rheumatoid arthritis.

Copy number variations (CNVs) have been implicated in human diseases. However, it remains unclear how they affect immune dysfunction and autoimmune diseases, including rheumatoid arthritis (RA). Here, we identified a novel leukocyte-specific protein 1 (LSP1) deletion variant for RA susceptibility located in 11p15.5. We replicated that the copy number of LSP1 gene is significantly lower in patients with RA, which correlates positively with LSP1 protein expression levels. Differentially expressed genes in Lsp1-deficient primary T cells represent cell motility and immune and cytokine responses. Functional assays demonstrated that LSP1, induced by T-cell receptor activation, negatively regulates T-cell migration by reducing ERK activation in vitro. In mice with T-cell-dependent chronic inflammation, loss of Lsp1 promotes migration of T cells into the target tissues as well as draining lymph nodes, exacerbating disease severity. Moreover, patients with RA show diminished expression of LSP1 in peripheral T cells with increased migratory capacity, suggesting that the defect in LSP1 signaling lowers the threshold for T-cell activation. To our knowledge, our work is the first to demonstrate how CNVs result in immune dysfunction and a disease phenotype. Particularly, our data highlight the importance of LSP1 CNVs and LSP1 insufficiency in the pathogenesis of RA and provide previously unidentified insights into the mechanisms underlying T-cell migration toward the inflamed synovium in RA.