41 research outputs found
Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases
Aims
The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them.
Methods and Results
We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analyzed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as “pathogenic” by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that 2 variants in KCNH2 and SCN5A, 4 variants in SCN10A, and 1 variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from “Uncertain significance” to “Likely pathogenic” in 6 probands.
Conclusions
Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD.
Translational Perspective
Whole-exome sequencing (WES) may be helpful in determining the causes of cardiac conduction system disease (CCSD), however, the identification of pathogenic variants remains a challenge. We performed WES of 23 probands diagnosed with early-onset CCSD, and identified 12 pathogenic or likely pathogenic variants in 11 of these probands (48%) according to the 2015 ACMG standards and guidelines. In this context, functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants, and SCN10A may be one of the major development factors in CCSD
The Fission Yeast Stress-Responsive MAPK Pathway Promotes Meiosis via the Phosphorylation of Pol II CTD in Response to Environmental and Feedback Cues
The RRM-type RNA-binding protein Mei2 is a master regulator of meiosis in fission yeast, in which it stabilizes meiosis-specific mRNAs by blocking their destruction. Artificial activation of Mei2 can provoke the entire meiotic process, and it is suspected that Mei2 may do more than the stabilization of meiosis-specific mRNAs. In our current study using a new screening system, we show that Mei2 genetically interacts with subunits of CTDK-I, which phosphorylates serine-2 residues on the C-terminal domain of RNA polymerase II (Pol II CTD). Phosphorylation of CTD Ser-2 is essential to enable the robust transcription of ste11, which encodes an HMG-type transcription factor that regulates the expression of mei2 and other genes necessary for sexual development. CTD Ser-2 phosphorylation increases under nitrogen starvation, and the stress-responsive MAP kinase pathway, mediated by Wis1 MAPKK and Sty1 MAPK, is critical for this stress response. Sty1 phosphorylates Lsk1, the catalytic subunit of CTDK-I. Furthermore, a feedback loop stemming from activated Mei2 to Win1 and Wis4 MAPKKKs operates in this pathway and eventually enhances CTD Ser-2 phosphorylation and ste11 transcription. Hence, in addition to starting meiosis, Mei2 functions to reinforce the commitment to it, once cells have entered this process. This study also demonstrates clearly that the stress-responsive MAP kinase pathway can modulates gene expression through phosphorylation of Pol II CTD
Immunohistochemical Profile for Unknown Primary Adenocarcinoma
BACKGROUND: Development of tailored treatment based on immunohistochemical profiles (IPs) of tumors for cancers of unknown primary is needed. METHODOLOGY/PRINCIPAL FINDINGS: We developed an algorithm based on primary known adenocarcinoma for testing sensitivity and specificity. Formalin-fixed paraffin-embedded tissue samples from 71 patients of unfavorable subsets of unknown primary adenocarcinoma were obtained. We examined 15 molecular markers using the algorithm incorporating these IPs and classified the tumours into 9 subsets based on the primary tumour site. The sensitivity and specificity of this algorithm were 80.3% and 97.6%, respectively. Apparent primary sites were lung in 17 patients, digestive organs in 13, gynecological organs in 9, prostate in 7, liver or kidney in 6, breast in 4, urothelial organ in 2, biliary tract and pancreatic profile in none, and unclassified in 13. The response rate to chemotherapy was highest for the gynecological IPs. Patients with gynecological or lung cancer IPs had longer median progression-free survival than those with others: 11.2 months for gynecological IPs (p<0.001) and 6.8 months for lung IPs (p = 0.05). Lung, digestive, prostate, and gynecological profiles were associated with significantly longer median survival time than the other profiles. Multivariate analysis confirmed that the IPs were independent prognostic factors for survival. CONCLUSIONS/SIGNIFICANCE: The IPs identified in this study can be used to further stratify patient prognosis for unfavorable subsets of unknown primary adenocarcinoma
CCN3 and bone marrow cells
CCN3 expression was observed in a broad variety of tissues from the early stage of development. However, a kind of loss of function in mice (CCN3 del VWC domain -/-) demonstrated mild abnormality, which indicates that CCN3 may not be critical for the normal embryogenesis as a single gene. The importance of CCN3 in bone marrow environment becomes to be recognized by the studies of hematopoietic stem cells and Chronic Myeloid Leukemia cells. CCN3 expression in bone marrow has been denied by several investigations, but we found CCN3 positive stromal and hematopoietic cells at bone extremities with a new antibody although they are a very few populations. We investigated the expression pattern of CCN3 in the cultured bone marrow derived mesenchymal stem cells and found its preference for osteogenic differentiation. From the analyses of in vitro experiment using an osteogenic mesenchymal stem cell line, Kusa-A1, we found that CCN3 downregulates osteogenesis by two different pathways; suppression of BMP and stimulation of Notch. Secreted CCN3 from Kusa cells inhibited the differentiation of osteoblasts in separate culture, which indicates the paracrine manner of CCN3 activity. CCN3 may also affect the extracellular environment of the niche for hematopoietic stem cells
Identification of Inappropriately Reprogrammed Genes by Large-Scale Transcriptome Analysis of Individual Cloned Mouse Blastocysts
Although cloned embryos generated by somatic/embryonic stem cell nuclear transfer (SECNT) certainly give rise to viable individuals, they can often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. In an effort to gain further insights into reprogramming and the properties of SECNT embryos, we performed a large-scale gene expression profiling of 87 single blastocysts using GeneChip microarrays. Sertoli cells, cumulus cells, and embryonic stem cells were used as donor cells. The gene expression profiles of 87 blastocysts were subjected to microarray analysis. Using principal component analysis and hierarchical clustering, the gene expression profiles were clearly classified into 3 clusters corresponding to the type of donor cell. The results revealed that each type of SECNT embryo had a unique gene expression profile that was strictly dependent upon the type of donor cells, although there was considerable variation among the individual profiles within each group. This suggests that the reprogramming process is distinct for embryos cloned from different types of donor cells. Furthermore, on the basis of the results of comparison analysis, we identified 35 genes that were inappropriately reprogrammed in most of the SECNT embryos; our findings demonstrated that some of these genes, such as Asz1, Xlr3a and App, were appropriately reprogrammed only in the embryos with a transcriptional profile that was the closest to that of the controls. Our findings provide a framework to further understand the reprogramming in SECNT embryos
Impact of tumor attachment to the pleura measured by a pretreatment CT image on outcome of stage I NSCLC treated with stereotactic body radiotherapy
The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination
Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination