RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.

Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.

Qualitative and quantitative analysis of MED12 c.887G>A causing both missense and splicing variants in X-linked Ohdo syndrome.

The phenotypes associated with MED12 pathogenic variants are diverse. Male patients usually have missense variants, but the effects of base substitutions on mRNA splicing have not been investigated. Here, we report a Japanese brother with intellectual disability, characteristic facial appearance with blepharophimosis, cleft palate, Fallot tetralogy, vesicoureteral reflux, and deafness. A known missense pathogenic variant was detected in MED12, NM_005120.3:c.887G>A p.(Arg296Gln), and X-linked Ohdo syndrome was diagnosed in combination with their phenotype. mRNA splicing of MED12 was evaluated qualitatively and quantitatively using long-range PCR-based targeted RNA sequencing (reverse transcribed long amplicon sequencing), and it was shown that this missense variant simultaneously causes aberrant splicing of the 42-bp in-frame deletion in exon 7, r.847_888del, which accounts for approximately 30% of the mRNAs in both siblings. The X chromosome inactivation study showed that the X chromosome carrying the mutant allele was 100% inactivated in the carrier mothers. mRNA level analysis is essential for the accurate interpretation of the effects of variants. In this case, the MED12 protein function may be reduced by more than just an amino acid substitution, resulting in the patients with the most severe phenotype of MED12-related syndrome in males.

Comparison of RNA-Sequencing Methods for Degraded RNA.

RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been developed. However, the relative advantage of each method for degraded RNA and low-input RNA, such as RNA samples collected in the field of clinical setting, has remained unknown. The Standard method of RNA-Seq captures mRNA by poly(A) capturing using Oligo dT beads, which is not suitable for degraded RNA. Here, we used three commercially available RNA-Seq library preparation kits (SMART-Seq, xGen Broad-range, and RamDA-Seq) using random primer instead of Oligo dT beads. To evaluate the performance of these methods, we compared the correlation, the number of detected expressing genes, and the expression levels with the Standard RNA-Seq method. Although the performance of RamDA-Seq was similar to that of Standard RNA-Seq, the performance for low-input RNA and degraded RNA has decreased. The performance of SMART-Seq was better than xGen and RamDA-Seq in low-input RNA and degraded RNA. Furthermore, the depletion of ribosomal RNA (rRNA) improved the performance of SMART-Seq and xGen due to increased expression levels. SMART-Seq with rRNA depletion has relative advantages for RNA-Seq using low-input and degraded RNA.

A comparison of mRNA sequencing (RNA-Seq) library preparation methods for transcriptome analysis.

BACKGROUND: mRNA sequencing is a powerful technique, which is used to investigate the transcriptome status of a gene of interest, such as its transcription level and splicing variants. Presently, several RNA sequencing (RNA-Seq) methods have been developed; however, the relative advantage of each method has remained unknown. Here we used three commercially available RNA-Seq library preparation kits; the traditional method (TruSeq), in addition to full-length double-stranded cDNA methods (SMARTer and TeloPrime) to investigate the advantages and disadvantages of these three approaches in transcriptome analysis. RESULTS: We observed that the number of expressed genes detected from the TeloPrime sequencing method was fewer than that obtained using the TruSeq and SMARTer. We also observed that the expression patterns between TruSeq and SMARTer correlated strongly. Alternatively, SMARTer and TeloPrime methods underestimated the expression of relatively long transcripts. Moreover, genes having low expression levels were undetected stochastically regardless of any three methods used. Furthermore, although TeloPrime detected a significantly higher proportion at the transcription start site (TSS), its coverage of the gene body was not uniform. SMARTer is proposed to be yielded for nonspecific genomic DNA amplification. In contrast, the detected splicing event number was highest in the TruSeq. The percent spliced in index (PSI) of the three methods was highly correlated. CONCLUSIONS: TruSeq detected transcripts and splicing events better than the other methods and measured expression levels of genes, in addition to splicing events accurately. However, although detected transcripts and splicing events in TeloPrime were fewer, the coverage at TSS was highest. Additionally, SMARTer was better than TeloPrime with regards to the detected number of transcripts and splicing events among the understudied full-length double-stranded cDNA methods. In conclusion, for short-read sequencing, TruSeq has relative advantages for use in transcriptome analysis.

Genotype and Phenotype Landscape of 283 Japanese Patients with Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by multiple dysplastic organ lesions and neuropsychiatric symptoms, caused by loss of function mutations in either TSC1 or TSC2. Genotype and phenotype analyses are conducted worldwide, but there have been few large-scale studies on Japanese patients, and there are still many unclear points. This study analyzed 283 Japanese patients with TSC (225 definite, 53 possible, and 5 genetic diagnoses). A total of 200 mutations (64 TSC1, 136 TSC2) were identified, of which 17 were mosaic mutations, 11 were large intragenic deletions, and four were splicing abnormalities due to deep intronic mutations. Several lesions and symptoms differed in prevalence and severity between TSC1 and TSC2 patients and were generally more severe in TSC2 patients. Moreover, TSC2 missense and in-frame mutations may attenuate skin and renal symptoms compared to other TSC2 mutations. Genetic testing revealed that approximately 20% of parents of a proband had mild TSC, which could have been missed. The patient demographics presented in this study revealed a high frequency of TSC1 patients and a low prevalence of epilepsy compared to global statistics. More patients with mild neuropsychiatric phenotypes were diagnosed in Japan, seemingly due to a higher utilization of brain imaging, and suggesting the possibility that a significant amount of mild TSC patients may not be correctly diagnosed worldwide.

The Detection of Immunity against WT1 and SMAD4P130L of EpCAM+ Cancer Cells in Malignant Pleural Effusion.

Malignant pleural effusion (MPE) provides a liquid tumor microenvironment model that includes cancer cells and immune cells. However, the characteristics of tumor antigen-specific CD8+ T cells have not been investigated in detail. Here, we analyzed MPE samples taken from a patient with pancreatic cancer who received a dendritic cell vaccine targeting Wilms' Tumor 1 (WT1) antigen over the disease course (two points at MPE1st and 2nd, two months after MPE1st). Epithelial cell adhesion molecule (EpCAM)+ cancer cells (PD-L1- or T cell immunoglobulin mucin-3, TIM-3-), both PD-1 or TIM-3 positive CD8+ T cells, and CD14+CD68+CD163+TIM-3+ macrophages increased from the MPE1st to MPE2nd. The ratio of WT1-specific cytotoxic lymphocytes (WT1-CTLs) to MPE CD8+ T cells and IFN-γ secretion of WT1-CTLs were reduced with disease progression. Coincidentally, the fraction of central memory T (TCM) of WT1-CTLs was decreased. On the other hand, CD8+ T cells in response to SMAD4P130L, which is homogeneously expressed in EpCAM+ cancer cells, were detected using in vitro expansion with the HLA-A*11:01 restrictive SVCVNLYH neoantigen. Furthermore, the CD8+ T cell response to SMAD4P130L was diminished following remarkably decreased numbers of CD8+ TCM in MPE samples. In conclusion, CD8+ T cells responding to WT1 or SMAD4P130L neoantigen expressed in EpCAM+ pancreatic cancer cells were detected in MPE. A tumor antigen-specific immune response would provide novel insight into the MPE microenvironment.

Incidence and Outcome of Aneurysmal Subarachnoid Hemorrhage: The Swiss Study on Subarachnoid Hemorrhage (Swiss SOS).

BACKGROUND AND PURPOSE: The purpose of this study was to assess nationwide incidence and outcomes of aneurysmal subarachnoid hemorrhage (aSAH). The Swiss SOS (Swiss Study on Subarachnoid Hemorrhage) was established in 2008 and offers the unique opportunity to provide this data from the point of care on a nationwide level. METHODS: All patients with confirmed aneurysmal subarachnoid hemorrhage admitted between January 1, 2009 and December 31, 2014, within Switzerland were recorded in a prospective registry. Incidence rates were calculated based on time-matched population data. Admission parameters and outcomes at discharge and at 1 year were recorded. RESULTS: We recorded data of 1787 consecutive patients. The incidence of aneurysmal subarachnoid hemorrhage in Switzerland was 3.7 per 100 000 persons/y. The number of female patients was 1170 (65.5%). With a follow-up rate of 91.3% at 1 year, 1042 patients (58.8%) led an independent life according to the modified Rankin Scale (0-2). About 1 in 10 patients survived in a dependent state (modified Rankin Scale, 3-5; n=185; 10.4%). Case fatality was 20.1% (n=356) at discharge and 22.1% (n=391) after 1 year. CONCLUSIONS: The current incidence of aneurysmal subarachnoid hemorrhage in Switzerland is lower than expected and an indication of a global trend toward decreasing admissions for ruptured intracranial aneurysms. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03245866.

Application of Combined Long Amplicon Sequencing (CoLAS) for Genetic Analysis of Neurofibromatosis Type 1: A Pilot Study.

Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.

Target-capture full-length double-strand cDNA sequencing for alternative splicing analysis.

Alternative splicing is a regulated process by which eukaryotic genes may produce diverse biological products. Defects in the process typically affect cellular function and can lead to disease. Next-generation sequencing (NGS) technologies have been developed to detect alternative splicing events; however, the alternative splicing events detected by standard RNA-Seq may or may not be derived from full-length RNA. The SMARTer method provides full-length double-strand cDNA synthesis, and the resulting gene expression patterns correlate strongly with standard RNA-Seq. However, it also yields non-specific genomic DNA amplification. We improved the SMARTer method by employing a target-capture full-length double-strand cDNA sequencing method. High-fidelity, full-length cDNA is generated by the SMARTer method, followed by target-specific capture with exon probes. The expression pattern observed with this SMARTer Capture method was highly correlated with the results of the original SMARTer method. The number and accuracy of the detected splicing events were increased by eliminating non-specific genomic DNA amplification by the SMARTer Capture. Compared to the original SMARTer method, the SMARTer Capture provided 4-fold greater detection of alternative splicing events at the same read number, and it took less than 1/100 of read number to detect the same number of splicing events. The percent splicing in index (PSI) of the SMARTer Capture is highly correlated with the PSI of the SMARTer. These results indicate that the SMARTer Capture represents an improvement of the SMARTer method to accurately characterize alternative splicing repertories in targeted genes without biases.

Context-dependent wiring of Sox2 regulatory networks for self-renewal of embryonic and trophoblast stem cells.

Sox2 is a transcription factor required for the maintenance of pluripotency. It also plays an essential role in different types of multipotent stem cells, raising the possibility that Sox2 governs the common stemness phenotype. Here we show that Sox2 is a critical downstream target of fibroblast growth factor (FGF) signaling, which mediates self-renewal of trophoblast stem cells (TSCs). Sustained expression of Sox2 together with Esrrb or Tfap2c can replace FGF dependency. By comparing genome-wide binding sites of Sox2 in embryonic stem cells (ESCs) and TSCs combined with inducible knockout systems, we found that, despite the common role in safeguarding the stem cell state, Sox2 regulates distinct sets of genes with unique functions in these two different yet developmentally related types of stem cells. Our findings provide insights into the functional versatility of transcription factors during embryogenesis, during which they can be recursively utilized in a variable manner within discrete network structures.