RefSeq Genes Research Articles

Skewed X-Chromosome Inactivation and Compensatory Upregulation of Escape Genes Precludes Major Clinical Symptoms in a Female With a Large Xq Deletion.

In mammalian females, X-chromosome inactivation (XCI) acts as a dosage compensation mechanism that equalizes X-linked genes expression between homo- and heterogametic sexes. However, approximately 12–23% of X-linked genes escape from XCI, being bi-allelic expressed. Herein, we report on genetic and functional data from an asymptomatic female of a Fragile X syndrome family, who harbors a large deletion on the X-chromosome. Array-CGH uncovered that the de novo, terminal, paternally originated 32 Mb deletion on Xq25-q28 spans 598 RefSeq genes, including escape and variable escape genes. Androgen receptor (AR) and retinitis pigmentosa 2 (RP2) methylation assays showed extreme skewed XCI ratios from both peripheral blood and buccal mucosa, silencing the abnormal X-chromosome. Surprisingly, transcriptome-wide analysis revealed that escape and variable escape genes spanning the deletion are mostly upregulated on the active X-chromosome, precluding major clinical/cognitive phenotypes in the female. Metaphase high count, hemizygosity concordance for microsatellite markers, and monoallelic expression of genes within the deletion suggest the absence of mosaicism in both blood and buccal mucosa. Taken together, our data suggest that an additional protective gene-by-gene mechanism occurs at the transcriptional level in the active X-chromosome to counterbalance detrimental phenotype effects of large Xq deletions.

Identification of novel mutations of ovarian cancer-related genes from RNA-sequencing data for Japanese epithelial ovarian cancer patients.

Ovarian cancer has the highest mortality rate among gynecological cancers. Gene mutations are involved in the carcinogenesis, metastasis, and therapeutic response in ovarian cancer. However, the variety and proportion of gene mutation is not fully analyzed in Japanese ovarian cancer patients, especially, in those with recurrent tumors. In the present study, RNA-sequencing was performed for 32 clinical ovarian specimens obtained from 24 Japanese patients (24 primary cancer specimens and 8 recurrent specimens paired with corresponding primary cancer specimens). Mutations in 24 primary specimens were analyzed by comparing the sequence data mapped on RefSeq genes with those in the public online databases BRCA Exchange, COSMIC, ClinVar, and cBioportal. Mutations were observed in TP53 in 16 specimens (67%), BRCA1 in 9 (38%), BRCA2 in 13 (54%), ARID1A in 3 (13%), PIK3CA in 2 (8%), KRAS in 1 (4%), PTEN in 1 (4%), and CTNNB1 in 1 (4%), excluding synonymous mutations. Among those identified muations, 13 of 14 mutations in TP53, 10 of 11 mutations of BRCA1, 10 of 23 mutation positions of BRCA2, none of 7 mutations of ARID1A, 1 mutation of PIK3CA, and 1 mutation of CTNNB1 were consistent with those reported in the public online databases; however, the other mutations identified were novel. Comparison between matched-paired specimens of primary and recurrent tumors revealed the changes of mutational status in expressed RNAs. RNA-sequencing-based mutation analysis will be useful to reveal ethnic differences of gene mutations in ovarian cancer and to understand the contribution of gene mutations to recurrence.

Abstract B070: LOCUS database enables rapid query and targeted selection of preclinical tumor models based on cancer genomics and pharmacologic response

Abstract Preclincal drug testing in oncology research has benefited from the development of thousands of tumors models—both in vitro and in vivo. Within BioDuro laboratories alone, there is a collection of >500 models, including traditional 2D human cell lines grown in vitro, primary 3D lines, cell-derived xenograft (CDX), patient derived xenograft (PDX) and syngeneic murine models (SYN). Identifying the most relevant models to support cancer drug discovery is a challenge. In an era of precision medicine, the need to understand tumor model characteristics, such as functional drug response profiles or genomic status, has become a critical path. In this report, we present LOCUS, a SQL backed database with graphical user interface, populated with functional pharmacologic response and genomic data for hundreds of tumor models including in vitro 2D, 3D, CDX, PDX and SYN. Models are readily searched by any number of conditions, including cancer type, model type, specific mutation, or target expression level. LOCUS combines relational databases for pharmacology and genomics data with scientific statistical models through a spring integrator. A scientific data centered domain architecture allows for pharmacogenomic queries and analysis. Pharmacogenomic data is pushed to dynamic scientific statistical models running on a python environment, the results of which are visualized to a Java EE UI Framework. All tumor models are genomically characterized, with searchable fields for single nucleotide variations, insertion deletions, and transcript expression levels. Extracted RNA was profiled using next generation sequencing performed on Illumina HiSeq using a paired end protocol. For xenograft samples, reads were first aligned and filtered by the mouse reference genome using the Bowtie2 algorithm. The remaining reads were aligned to the GRCh38 human reference genome using the MapSplice2 algorithm. Transcript- and Gene-level expression were determined by counting the number of sequence alignments per exon according to the RefSeq gene model, and computing Fragments Per Killobase Million (FPKM) and Transcripts Per Million (TPM) values using best practices. Opossum software was used to further prepare alignments produced by MapSplice2 algorithm for small variant calling. The Platypus variant caller was used to identify single nucleotide variant, small insertion, and small deletion events. The variation events were annotated using SnpEff and custom perl scripts to overlay known somatic mutations with oncogenic significance. LOCUS provides users with rapid access to hundreds of pharmacology and genomic data sets that enable rational selection of tumor models for preclinical oncology drug testing. The database and structure are readily scaled, supporting future growth of thousands of more tumor models and characterization. Citation Format: Armand Amin, Sandeep Sanga, Shanshan Gan, Ruyi Li, Todd Blewett, Zhuyao Wang, Thomas B Broudy. LOCUS database enables rapid query and targeted selection of preclinical tumor models based on cancer genomics and pharmacologic response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B070. doi:10.1158/1535-7163.TARG-19-B070

Full-length transcriptome reconstruction reveals a large diversity of RNA and protein isoforms in rat hippocampus

Gene annotation is a critical resource in genomics research. Many computational approaches have been developed to assemble transcriptomes based on high-throughput short-read sequencing, however, only with limited accuracy. Here, we combine next-generation and third-generation sequencing to reconstruct a full-length transcriptome in the rat hippocampus, which is further validated using independent 5´ and 3´-end profiling approaches. In total, we detect 28,268 full-length transcripts (FLTs), covering 6,380 RefSeq genes and 849 unannotated loci. Based on these FLTs, we discover co-occurring alternative RNA processing events. Integrating with polysome profiling and ribosome footprinting data, we predict isoform-specific translational status and reconstruct an open reading frame (ORF)-eome. Notably, a high proportion of the predicted ORFs are validated by mass spectrometry-based proteomics. Moreover, we identify isoforms with subcellular localization pattern in neurons. Collectively, our data advance our knowledge of RNA and protein isoform diversity in the rat brain and provide a rich resource for functional studies.

1369P - Genes involved in DNA replication, chromatin remodeling and cell cycle as potential biomarkers for therapy outcome to immune therapy in patients with metastatic cutaneous malignant melanoma

BackgroundImmune checkpoint inhibitors (ICI) have improved clinical outcome for many patients with advanced cutaneous malignant melanoma (CMM) during the last decade. The magnitude and duration of response vary considerably between patients. Predictive biomarkers to identify patients that will benefit from treatment can increase efficacy, diminish side effects and costs. Our aim is to identify predictive biomarkers in tumors samples from patients with advanced CMM receiving ICI. MethodsPatients with advanced CMM at Karolinska University Hospital, Stockholm, starting therapy with ICI were invited to participate in our study. After signing informed consent a pre-treatment fine needle or core biopsy was taken from an accessible metastasis. RNA was extracted to perform targeted RNA sequencing using the Ion AmpliSeq Transcriptome Human Gene Expression Kit for RefSeq genes. Partek Genomics Suite® software was applied to find differentially expressed genes and correlate the data with therapy response and progression free survival (PFS). ResultsNineteen patients were included between September2013 and August2017, 8 female and 11 male. The median age was 70 years old (range 49 – 84). All patients had metastatic disease (13 M1c, 4 M1b, 2 M1a). ICI was first-line treatment for 16 patients. Nivolumab or pembrolizumab was given to 17 patients and two received ipilimumab. Five patients had partial response, 3 stable disease and 7 complete response whereas 4 had progressive diasease. Median PFS was 10 months (range 1,2 – 62 months, 6 patients still responding). High expression of a subset of genes playing a role for DNA replication, genes involved in chromatin remodeling and cell cycle were significantly associated with shorter PFS. The correlation between low expression of interferon gamma signature genes and poorer treatment outcome was confirmed in our study. ConclusionsOur findings suggest that genes involved in the regulation of DNA replication, chromatin remodeling and cell cycle may influence the long-term response to ICI. Legal entity responsible for the studyKarolinska University Hospital and Karolinska Institute. FundingThe Swedish Cancer Society, the Cancer Research Funds of Radiumhemmet and Knut and Alice Wallenberg foundation. We aknowledge support of the Science for Life Laboratory, National Genomics Infrastructure (NGI)/Uppsala, Genome Center and UPPMAX for providing assistance in massive parallel sequencing and computational infrastructure (work funded by RFI/VR and Scilife, Sweden). DisclosureAll authors have declared no conflicts of interest.

Considering smoking status, coexpression network analysis of non-small cell lung cancer at different cancer stages, exhibits important genes and pathways.

Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer among smokers, nonsmokers, women, and young individuals. Tobacco smoking and different stages of the NSCLC have important roles in cancer evolution and require different treatments. Existence of poorly effective therapeutic options for the NSCLC brings special attention to targeted therapies by considering genetic alterations. In this study, we used RNA-Seq data to compare expression levels of RefSeq genes and to find some genes with similar expression levels. We utilized the "Weighted Gene Co-expression Network Analysis" method for three different datasets to create coexpressed genetic modules having relations with the smoking status and different stages of the NSCLC. Our results indicate seven important genetic modules having important associations with the smoking status and cancer stages. Based on investigated genetic modules and their biological explanation, we then identified 13 newly candidate genes and 7 novel transcription factors in association with the NSCLC, the smoking status, and cancer stages. We then examined those results using other datasets and explained our results biologically to illustrate some important genes in relation with the smoking status and metastatic stage of the NSCLC that can bring some crucial information about cancer evolution. Our genetic findings also can be used as some therapeutic targets for different clinical conditions of the NSCLC.

Microdeletions excluding YWHAE and PAFAH1B1 cause a unique leukoencephalopathy: further delineation of the 17p13.3 microdeletion spectrum

PurposeBrain malformations caused by 17p13.3 deletions include lissencephaly with deletions of the larger Miller–Dieker syndrome region or smaller deletions of only PAFAH1B1, white matter changes, and a distinct syndrome due to deletions including YWHAE and CRK but sparing PAFAH1B1. We sought to understand the significance of 17p13.3 deletions between the YWHAE/CRK and PAFAH1B1 loci. MethodsWe analyzed the clinical features of six individuals from five families with 17p13.3 deletions between and not including YWHAE/CRK and PAFAH1B1 identified among individuals undergoing clinical chromosomal microarray testing or research genome sequencing. ResultsFive individuals from four families had multifocal white matter lesions while a sixth had a normal magnetic resonance image. A combination of our individuals and a review of those in the literature with white matter changes and deletions in this chromosomal region narrows the overlapping region for this brain phenotype to ~345kb, including 11 RefSeq genes, with RTN4RL1 haploinsufficiency as the best candidate for causing this phenotype. ConclusionWhile previous literature has hypothesized dysmorphic features and white matter changes related to YWHAE, our cohort contributes evidence to the presence of additional genetic changes within 17p13.3 required for proper brain development.

Abstract 2472: LOCUS: Queryable database of cancer genomics and pharmacologic response enables rapid selection of in vitro and in vivo preclinical tumor models

Abstract Preclincal drug testing in oncology research has benefited from the development of thousands of tumors models—both in vitro and in vivo. Within BioDuro laboratories alone, there is a collection of >500 models, including traditional 2D human cell lines grown in vitro, primary 3D lines, cell-derived xenograft (CDX), patient derived xenograft (PDX) and syngeneic murine models (SYN). Identifying the most relevant models to support cancer drug discovery is a challenge. In an era of precision medicine, the need to understand tumor model characteristics, such as functional drug response profiles or genomic status, has become a critical path. In this report, we present LOCUS, a SQL backed database with graphical user interface, populated with functional pharmacologic response and genomic data for hundreds of tumor models including in vitro 2D, 3D, CDX, PDX and SYN. Models are readily searched by any number of conditions, including cancer type, model type, specific mutation, or target expression level. LOCUS combines relational databases for pharmacology and genomics data with scientific statistical models through a spring integrator. A scientific data centered domain architecture allows for pharmacogenomic queries and analysis. Pharmacogenomic data is pushed to dynamic scientific statistical models running on a python environment, the results of which are visualized to a Java EE UI Framework. All tumor models are genomically characterized, with searchable fields for single nucleotide variations, insertion deletions, and transcript expression levels. Extracted RNA was profiled using next generation sequencing performed on Illumina HiSeq using a paired end protocol. For xenograft samples, reads were first aligned and filtered by the mouse reference genome using the Bowtie2 algorithm. The remaining reads were aligned to the GRCh38 human reference genome using the MapSplice2 algorithm. Transcript- and Gene-level expression were determined by counting the number of sequence alignments per exon according to the RefSeq gene model, and computing Fragments Per Killobase Million (FPKM) and Transcripts Per Million (TPM) values using best practices. Opossum software was used to further prepare alignments produced by MapSplice2 algorithm for small variant calling. The Platypus variant caller was used to identify single nucleotide variant, small insertion, and small deletion events. The variation events were annotated using SnpEff and custom perl scripts to overlay known somatic mutations with oncogenic significance. LOCUS provides users with rapid access to hundreds of pharmacology and genomic data sets that enable rational selection of tumor models for preclinical oncology drug testing. The database and structure are readily scaled, supporting future growth of thousands of more tumor models and characterization. Citation Format: Armand Amin, Sandeep Sanga, Shanshan Gan, Yong Hu, Thomas Broudy. LOCUS: Queryable database of cancer genomics and pharmacologic response enables rapid selection of in vitro and in vivo preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2472.

Epigenetic signatures of Werner syndrome occur early in life and are distinct from normal epigenetic aging processes.

Werner Syndrome (WS) is an adult‐onset segmental progeroid syndrome. Bisulfite pyrosequencing of repetitive DNA families revealed comparable blood DNA methylation levels between classical (18 WRN‐mutant) or atypical WS (3 LMNA‐mutant and 3 POLD1‐mutant) patients and age‐ and sex‐matched controls. WS was not associated with either age‐related accelerated global losses of ALU, LINE1, and α‐satellite DNA methylations or gains of rDNA methylation. Single CpG methylation was analyzed with Infinium MethylationEPIC arrays. In a correspondence analysis, atypical WS samples clustered together with the controls and were clearly separated from classical WS, consistent with distinct epigenetic pathologies. In classical WS, we identified 659 differentially methylated regions (DMRs) comprising 3,656 CpG sites and 613 RefSeq genes. The top DMR was located in the HOXA4 promoter. Additional DMR genes included LMNA, POLD1, and 132 genes which have been reported to be differentially expressed in WRN‐mutant/depleted cells. DMRs were enriched in genes with molecular functions linked to transcription factor activity and sequence‐specific DNA binding to promoters transcribed by RNA polymerase II. We propose that transcriptional misregulation of downstream genes by the absence of WRN protein contributes to the variable premature aging phenotypes of WS. There were no CpG sites showing significant differences in DNA methylation changes with age between WS patients and controls. Genes with both WS‐ and age‐related methylation changes exhibited a constant offset of methylation between WRN‐mutant patients and controls across the entire analyzed age range. WS‐specific epigenetic signatures occur early in life and do not simply reflect an acceleration of normal epigenetic aging processes.

Genomic Analysis Reveals Specific Patterns of Homozygosity and Heterozygosity in Inbred Pigs.

Simple SummaryThe inbred strain of miniature pig is an ideal model for biomedical research due to its high level of homozygosity. Our results from multidimensional scaling, admixture, and phylogenetic analyses indicated that the inbred Wuzhishan pig (WZSP), with its unique genetic properties, can be utilized as a novel genetic resource for pig genome studies. Inbreeding depression and run of homozygosity (ROH) analyses revealed an average of 61 and 12 ROH regions in the inbred and non-inbred genomes of WZSPs, respectively. ROH number, length, and distribution across generations indicate the impacts of recombination and demography on ROHs in these WZSPs. Finally, we detected 56 single nucleotide polymorphisms (SNPs) showing constant heterozygosity with heterozygosity (He) = 1 across six generations in inbred pigs, while only one was found in the non-inbred population. Among these SNPs, we observed nine SNPs located in swine RefSeq genes, which were found to be involved in signaling and immune processes. Together, our findings indicate that the inbred-specific pattern of homozygosity and heterozygosity in inbred pigs can offer valuable insights for elucidating the mechanisms of inbreeding in farm animals.The inbred strain of miniature pig is an ideal model for biomedical research due to its high level of homozygosity. In this study, we investigated genetic diversity, relatedness, homozygosity, and heterozygosity using the Porcine SNP60K BeadChip in both inbred and non-inbred Wuzhishan pigs (WZSPs). Our results from multidimensional scaling, admixture, and phylogenetic analyses indicated that the inbred WZSP, with its unique genetic properties, can be utilized as a novel genetic resource for pig genome studies. Inbreeding depression and run of homozygosity (ROH) analyses revealed an average of 61 and 12 ROH regions in the inbred and non-inbred genomes of WZSPs, respectively. By investigating ROH number, length, and distribution across generations, we further briefly studied the impacts of recombination and demography on ROH in these WZSPs. Finally, we explored the SNPs with higher heterozygosity across generations and their potential functional implications in the inbred WZSP. We detected 56 SNPs showing constant heterozygosity with He = 1 across six generations in inbred pigs, while only one was found in the non-inbred population. Among these SNPs, we observed nine SNPs located in swine RefSeq genes, which were found to be involved in signaling and immune processes. Together, our findings indicate that the inbred-specific pattern of homozygosity and heterozygosity in inbred pigs can offer valuable insights for elucidating the mechanisms of inbreeding in farm animals.

Whole genome sequencing analysis of horse populations inhabiting the Korean Peninsula and Przewalski's horse.

The Jeju horse is an indigenous horse breed in Korea. However, there is a severe lack of genomic studies on Korean horse breeds. The objective of this study was to report genomic characteristics of domestic horse populations that inhabit South Korea (Jeju, Jeju crossbred, and Thoroughbred) and a wild horse breed (Przewalski's horse). Using the equine reference genome assembly (EquCab 2.0), more than ~ 6.5billion sequence reads were successfully mapped, which generated an average of 40.87-fold coverage throughout the genome. Using these data, we detected a total of 12.88million SNPs, of which 73.7% were found to be novel. All the detected SNPs were deeply annotated to retrieve SNPs in gene regions using the RefSeq and Ensemble gene sets. Approximately 27% of the total SNPs were located within genes, whereas the remaining 73% were found in intergenic regions. Using 129,776 coding SNPs, we retrieved a total of 49,171 nonsynonymous SNPs in 12,351 genes. Furthermore, we identified a total of 10,770 deleterious nonsynonymous SNPs which are predicted to affect protein structure or function. We showed numerous genomic variants from domestic and wild horse breeds. These results provide a valuable resource for further studies on functions of SNP-containing genes, and can aid in determining the molecular basis underlying variation in economically important traits of horses.

Neonate with 10q Interstitial Deletion within the Long Arm of Chromosome 10- A Case Report and Literature Review

Introduction: Partial deletion of distal chromosome 10q was first reported in 1978 by Lewandowski1. Interstitial deletions within bands 10q25e10q26.3 are rare. Seven such cases were reported so far2. Patient Information: A term AGA male newborn was delivered at our perinatal center with antenatal diagnosis of unbalanced translocation of chromosomes 10 and 12, and fetal cleft lip and cleft palate. Blood was sent for chromosome analysis using GTG banding method. Baby had facial dysmorphism, left cleft lip, bilateral cleft of soft and hard palate, intact nasal septum, normal ears and micrognathus. Abdominal ultrasound showed absence of right testis in inguinal canal and abdomen (anorchia). Hospital course was unremarkable except for feeding problems requiring feeding team, plastic surgery planned at 2- 3 months of age, and taping of the cleft lip. He went home on day 7. Conclusion: Here, we reported an extremely rare case of a male newborn with an interstitial deletion within the long arm of chromosome 10 between bands 10q25.1 and 10q26.1, with dysmorphic features, along with a few unreported associations (atypical Pierre-Robin sequence and bilateral dorsal horn ventriculomegaly). We added a comprehensive review of literature on chromosome 10q deletions to the case report. We also listed clinical implications of 71 RefSeq and OMIM genes noted in that ~13.3Mb 10q deletion in the Appendix. Earlier detection of both common and rare chromosomal- genetic abnormalities might prepare the family and health care team to plan optimal care to the mother, and baby.

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform. Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

DNA methylation subpatterns at distinct regulatory regions in human early embryos.

DNA methylation has been investigated for many years, but recent technologies have allowed for single-cell- and single-base-resolution DNA methylation datasets and more accurate assessment of DNA methylation dynamics at the key genomic regions that regulate gene expression in human early embryonic development. In this study, the region from upstream 20 kb to downstream 20 kb of RefSeq gene was selected and divided into 12 distinct regions (up20, up10, up5, up2, 5'UTR, exon, intron, 3'UTR, down2, down5, down10 and down20). The candidate promoter region (TSS ± 2 kb) was further divided into 20 consecutive subregions, which were termed ‘bins’. The DNA methylation dynamics of these regions were systematically analysed along with their effects on gene expression in human early embryos. The dynamic DNA methylation subpatterns at the distinct genomic regions with a focus on promoter regions were mapped. For the 12 distinct genomic regions, up2 and 5'UTR had the lowest DNA methylation levels, and their methylation dynamics were different with other regions. The region 3'UTR had the highest DNA methylation levels, and the correlation analysis with gene expression proved that it was a feature of transcribed genes. For the 20 bins in promoter region, the CpG densities showed a normal distribution pattern, and the trend of the methylated CpG counts was inverse with the DNA methylation levels, especially for the bin 1 (downstream 200 bp of the TSS). Through the correlation analysis between DNA methylation and gene expression, the current study finally revealed that the region bin −4 to 6 (800 bp upstream to 1200 bp downstream of the TSS) was the best candidate for the promoter region in human early embryos, and bin 1 was the putative key regulator of gene activity. This study provided a global and high-resolution view of DNA methylation subpatterns at the distinct genomic regions in human early embryos.

Novel deletion of 6p21.31p21.1 associated with laryngeal cleft, developmental delay, dysmorphic features and vascular anomaly

Interstitial deletions involving chromosome region 6p21.31p21.2 have not been previously reported in the literature. Here, we present a 2 year old girl with global developmental delay, severe speech delay, dysmorphic features, laryngeal cleft, anterior descending aorta that occluded the left main bronchus and a novel de novo deletion of chromosome 6: arr[hg19] 6p21.31p21.2 (35462950–36725083)x1. The deletion, which was diagnosed by array comparative genomic hybridization and further confirmed with fluorescence in situ hybridization, was approximately 1.26 Mb and contained 28 RefSeq genes. The deleted region includes 24 protein coding genes and 4 non-coding genes. This represents a novel microdeletion that has not been previously reported in the literature.

TSPYL2 Regulates the Expression of EZH2 Target Genes in Neurons

Testis-specific protein, Y-encoded-like 2 (TSPYL2) is an X-linked gene in the locus for several neurodevelopmental disorders. We have previously shown that Tspyl2 knockout mice had impaired learning and sensorimotor gating, and TSPYL2 facilitates the expression of Grin2a and Grin2b through interaction with CREB-binding protein. To identify other genes regulated by TSPYL2, here, we showed that Tspyl2 knockout mice had an increased level of H3K27 trimethylation (H3K27me3) in the hippocampus, and TSPYL2 interacted with the H3K27 methyltransferase enhancer of zeste 2 (EZH2). We performed chromatin immunoprecipitation (ChIP)-sequencing in primary hippocampal neurons and divided all Refseq genes by k-mean clustering into four clusters from highest level of H3K27me3 to unmarked. We confirmed that mutant neurons had an increased level of H3K27me3 in cluster 1 genes, which consist of known EZH2 target genes important in development. We detected significantly reduced expression of genes including Gbx2 and Prss16 from cluster 1 and Acvrl1, Bdnf, Egr3, Grin2c, and Igf1 from cluster 2 in the mutant. In support of a dynamic role of EZH2 in repressing marked synaptic genes, the specific EZH2 inhibitor GSK126 significantly upregulated, while the demethylase inhibitor GSKJ4 downregulated the expression of Egr3 and Grin2c. GSK126 also upregulated the expression of Bdnf in mutant primary neurons. Finally, ChIP showed that hemagglutinin-tagged TSPYL2 co-existed with EZH2 in target promoters in neuroblastoma cells. Taken together, our data suggest that TSPYL2 is recruited to promoters of specific EZH2 target genes in neurons, and enhances their expression for proper neuronal maturation and function.

Single cell transcriptome analysis of MCF-7 reveals consistently and inconsistently expressed gene groups each associated with distinct cellular localization and functions.

Single cell transcriptome (SCT) analysis provides superior resolution to illustrate tumor cell heterogeneity for clinical implications. We characterized four SCTs of MCF-7 using 143 housekeeping genes (HKGs) as control, of which lactate dehydrogenase B (LDHB) expression is silenced. These SCT libraries mapped to 11,423, 11,486, 10,380, and 11,306 RefSeq genes (UCSC), respectively. High consistency in HKG expression levels across all four SCTs, along with transcriptional silencing of LDHB, was observed, suggesting a high sensitivity and reproducibility of the SCT analysis. Cross-library comparison on expression levels by scatter plotting revealed a linear correlation and an 83–94% overlap in transcript isoforms and expressed genes were also observed. To gain insight of transcriptional diversity among the SCTs, expressed genes were split into consistently expressed (CE) (expressed in all SCTs) and inconsistently expressed (IE) (expressed in some but not all SCTs) genes for further characterization, along with the 142 expressed HKGs as a reference. Distinct transcriptional strengths were found among these groups, with averages of 1,612.0, 88.0 and 1.2 FPKM for HKGs, CE and IE, respectively. Comparison between CE and IE groups further indicated that expressions of CE genes vary more significantly than that of IE genes. Gene Ontology analysis indicated that proteins encoded by CE genes are mainly involved in fundamental intracellular activities, while proteins encoded by IE genes are mainly for extracellular activities, especially acting as receptors or ion channels. The diversified gene expressions, especially for those encoded by IE genes, may contribute to cancer drug resistance.

Stigmasterol upregulates immediate early genes and promotes neuronal cytoarchitecture in primary hippocampal neurons as revealed by transcriptome analysis

BackgroundThe hippocampus is a vulnerable brain region that is implicated in learning and memory impairment by two pathophysiological features, that is, neurite regression and synaptic dysfunction, and stigmasterol (ST), a cholesterol-equivalent phytosterol, is known to facilitate neuromodulatory effects. PurposeTo investigate the neuromodulatory effects of ST on the development of central nervous system neurons and the molecular bases of these effects in primary hippocampal neurons. MethodsRat embryonic (E18-19) brain neurons were cultured in the absence or presence of ST (75 µM). Neuritogenic activities of ST were evident by increases in various morphometric parameters. To identify underlying affected genes, total RNA was isolated on day in vitro 12 (DIV 12) and mRNA high throughput sequencing (mRNA-Seq) was performed. Affected key genes for neuronal development were identified using bioinformatics tools and their upregulations were confirmed by immunocytochemistry. ResultsAmong the differentially expressed 17,337 RefSeq genes, 445 genes (up/down 293/157) passed the p-value < 0.05 criterion, 52 genes (up/down; 37/13) had a p-value < 0.05 and a false discovery rate (FDR) q-value of < 0.2, and 24 genes (up/down; 20/4) passed the more stringent criterion of both p < 0.05 and q < 0.05. After applying a stringent FDR q-value cutoff of < 0.2, it was found ST induced many immediate early genes (IEGs), and that a major proportion of upregulated genes were related to central nervous system (CNS) development (neurite outgrowth or synaptic transmission). In a Venn diagram for CNS development Gene Ontologies (GOs) (i.e., axon development, dendrite development, modulation of synaptic transmission), Reln emerged as a central player in these processes, and highly interconnected ‘hub’ genes, including Dcx, Egr1, Ntrk2, and Slc24a2, were revealed by gene co-expression networks. Finally, transcriptomic data was confirmed by immunocytochemistry of primary hippocampal neurons. ConclusionThe study indicates that ST upregulates genes for neuritogenesis and synaptogenesis, and suggests ST be viewed as a potential resource for improving brain functions.

Transcriptional interference by small transcripts in proximal promoter regions.

Proximal promoter regions (PPR) are heavily transcribed yielding different types of small RNAs. The act of transcription within PPRs might regulate downstream gene expression via transcriptional interference (TI). For analysis, we investigated capped and polyadenylated small RNA transcripts within PPRs of human RefSeq genes in eight different cell lines. Transcripts of our datasets overlapped with experimentally determined transcription factor binding sites (TFBS). For TFBSs intersected by these small RNA transcripts, we established negative correlation of sRNA expression levels and transcription factor (TF) DNA binding affinities; suggesting that the transcripts acted via TI. Accordingly, datasets were designated as TFbiTrs (TF-binding interfering transcripts). Expression of most TFbiTrs was restricted to certain cell lines. This facilitated the analysis of effects related to TFbiTr expression for the same RefSeq genes across cell lines. We consistently uncovered higher relative TF/DNA binding affinities and concomitantly higher expression levels for RefSeq genes in the absence of TFbiTrs. Analysis of corresponding chromatin landscapes supported these results. ChIA-PET revealed the participation of distal enhancers in TFbiTr transcription. Enhancers regulating TFbiTrs, in effect, act as repressors for corresponding downstream RefSeq genes. We demonstrate the significant impact of TI on gene expression using selected small RNA datasets.