Project description:We identified a histone H3.3 sub-variant, H3mm7, to be specifically expressed in skeletal muscle satellite cells. H3mm7 knockout mice demonstrated an essential role of H3mm7 in skeletal muscle regeneration. Chromatin analysis revealed that H3mm7 facilitates transcription by forming an open chromatin structure around promoter regions including those of myogenic genes.
Project description:Background: Genetic testing of a child with persistent EBV viremia identified a novel variant c.1279dupT (p.*427Leuext*42) in interferon regulatory factor 8 (IRF8). The same variant was also found in his mother, who shortly thereafter was diagnosed with an HPV+ tumor. The maternal grandparents lacked this variant. Objective: We sought to examine the pathogenicity of the identified IRF8 variant and its phenotypic and functional characteristics in the proband and his mother. Methods: Immune cell enumeration, immunophenotypic and functional flow cytometry, NK cell cytotoxicity, MALDI-TOF mass spectrometry, TCRVb spectratyping, RNA sequencing, OLink proteomics, Sanger sequencing, Immunoblotting, Molecular cloning, Immunofluorescence microscopy and Image analysis were used in this study.
Project description:Background: Cerebral Cavernous Malformation (CCM) is a severe neurovascular disease that shows autosomal dominant inheritance in families. The disease shows wide phenotypic variability, with some patients developing vascular lesions in infancy and others never experiencing symptoms. The majority of familial CCM cases are caused by mutations in KRIT1, CCM2, or PDCD10. However, 5–15% of familial cases and ~40% of sporadic cases with multiple lesions do not exhibit variants in these genes. It remains unknown whether non-coding variants or variants in novel genes contribute to genotype-negative disease or phenotypic variability. Methods: We performed whole genome sequencing (WGS) on a family with two siblings with late-onset CCM and an unaffected third sibling. Next we identified potential causal variants that segregated with disease, were rare (allele frequency <0.001), and were either predicted loss-of-function or in CCM pathway genes. Candidate variants were assessed using in silico splice prediction tools and functional assays to evaluate their effects on protein structure and gene expression. Results: The prioritized causal variants identified with WGS in this family were in the KRIT1 and PIK3CA genes. First we prioritized a single base pair substitution in intron 5 of KRIT1 (c.262+115A>G) predicted to affect splicing. We found the variant leads to inclusion of a novel 129-bp exon containing multiple stop codons in KRIT1 mRNA, likely causing loss of protein function via nonsense-mediated decay or generation of nonfunctional protein. This variant was absent from population databases (ClinVar) and segregated with disease in the family. A second potential disease-relevant variant was in intron 1 of PIK3CA (c.-77+1G>A). Prime editing of this variant in a human aortic endothelial cell line resulted in a 1.5-fold increase in PIK3CA expression. Discussion: This is the first report of WGS identifying causal non-coding single base pair variants in a case of genotype-negative familial CCM. Our findings highlight the diagnostic limitations of exon-focused sequencing and support the application of WGS for variant-negative cases. In this case there is a possible contribution of two non-coding variants that have functional effects on known CCM risk genes. Expanding genetic screening to noncoding regions may improve diagnostic yield, genetic counseling, and CCM risk stratification.
Project description:Analysis of Beat AML sequencing data revealed a recurrent frameshift variant (R939Pfs*Ter36) in Zinc Finger 687 (ZNF687) that was observed in ~1% of patients and has not been previously described. ZNF687 encodes a transcription factor containing DNA binding C2H2 zinc finger and has been studied in select other malignancies, including Paget’s disease of bone that is associated with giant cell tumor (PDB/GCT) and hepatocellular carcinoma. It is part of a network with ZMYND8 and two additional zinc finger proteins, ZNF592 and ZNF532, that associate with H3K4 demethylation machinery and regulates gene transcription. The R939Pfs*Ter36 frameshift induced overexpression of ZNF687 compared with wild-type, and the overexpressed protein was mislocalized to the cytoplasm. We further performed RNA sequencing on HEK293 cells expressing the frameshift variant, wild-type, or a previously identified variant (P937R) that has been reported as a germline ZNF687 variant characterized to predispose to PDB/GCT. We observed the R939Pfs*Ter36 frameshift to induce activation of oxidative phosphorylation, inactivation of histone methylation machinery, and significant downregulation of lysine methyltransferase 2D (KMT2D) compared with wild-type and P937R. Overall, these findings suggest that ZNF687 R939Pfs*Ter36 plays an important role in AML pathogenesis, which could aid in diagnostic precision, and may impact therapeutic responses.
Project description:In this study, we recruited a patient with Hereditary spherocytosis (HS) detected to have a novel heterozygous variant in the SPTB in the proband. Sanger sequencing of variant alleles and haplotype linkage analysis were performed simultaneously. Five embryos were identified with one heterozygous and four not carrying the SPTB variant. Single-cell amplification and whole genome sequencing showed that three embryos had varying degrees of trisomy mosaicism.
Project description:Probing the rice genome for novel histone coding genes revealed a genera specific, histone H4 variant with several variations in N-terminal tail sequence motifs, expressing in a tissue-specific manner. Genetic perturbation of the H4 variant in rice led to defects in growth and fertility. Probing the chromatin binding sites of the H4 variant with a custom antibody revealed its binding at specific genes and its knockout led to mis-expression of stress-associated genes. Stress-challenged plants showed loss of the H4 variant and re-distribution of other H4 linked histone marks. In vitro reconstituted rice nucleosomes with H4 variant histones displayed atypical properties in terms of stability, intra-nucleosome binding and structure, when compared to the canonical nucleosomes.
Project description:Probing the rice genome for novel histone coding genes revealed a genera specific, histone H4 variant with several variations in N-terminal tail sequence motifs, expressing in a tissue-specific manner. Genetic perturbation of the H4 variant in rice led to defects in growth and fertility. Probing the chromatin binding sites of the H4 variant with a custom antibody revealed its binding at specific genes and its knockout led to mis-expression of stress-associated genes. Stress-challenged plants showed loss of the H4 variant and re-distribution of other H4 linked histone marks. In vitro reconstituted rice nucleosomes with H4 variant histones displayed atypical properties in terms of stability, intra-nucleosome binding and structure, when compared to the canonical nucleosomes.
Project description:Probing the rice genome for novel histone coding genes revealed a genera specific, histone H4 variant with several variations in N-terminal tail sequence motifs, expressing in a tissue-specific manner. Genetic perturbation of the H4 variant in rice led to defects in growth and fertility. Probing the chromatin binding sites of the H4 variant with a custom antibody revealed its binding at specific genes and its knockout led to mis-expression of stress-associated genes. Stress-challenged plants showed loss of the H4 variant and re-distribution of other H4 linked histone marks. In vitro reconstituted rice nucleosomes with H4 variant histones displayed atypical properties in terms of stability, intra-nucleosome binding and structure, when compared to the canonical nucleosomes.
Project description:In a meta-analysis of three GWASs in the Japanese population, we identified a novel locus at 16p12.3 significantly associated with pancreatic cancer susceptibility. A series of experiments were performed to explore the functional impact of the nonsynonymous lead SNP rs78193826 of the GP2 gene at 16p12.3 on global gene expression. Based on CRISPR-Cas9-induced mutation, RNA-seq, and GSEA analyses, we demonstrated that the functional relevance of rs78193826 may involve modulation of KRAS activity.