Project description:We performed bulk RNA-seq analysis for CRISPR-Cpf1 mutated H1 cells that were differentiated into pancreatic cells. Homozygous mutant was compared to control cells (RFX6-/- vs RFX6+/+) at stage 3 (PF) and stage 5 (EP), and heterozygous mutant was compared to control cells (RFX6-/+ vs RFX6+/+) at stage 7 (SC-islets).
Project description:LATS1/2 are canonical Hippo signaling pathway components. Our genome-wide screen indicated a synthetic viable effect of Hippo pathway inhibition in ATM-depleted human embryonic and neural progenitor cells. This experiment was designed in order to get mechanistic insights regarding the molecular effect of Hippo pathway inhibition on ATM-knockout cells. Such chemical inhibition could potentially be used as a means to impede Ataxia-Telangiectasia-related neurodegeneration. Experimental procedure: 2 clones of ATM-knockout h-pES10 cells were plated on 6 well plates with MEFs feeder layer. 1 d after plating, medium was replaced with standard medium (as control) or medium containing 10 µM of LATS1/2 inhibitor, TRULI (Lats-IN-1) for 24 h. Cells were then harvested, total RNA was extracted, libraries for RNA sequencing were generated and sequenced. Total reads were mapped to human GRCh38 reference genome, and to mouse GRCm38 using STAR package. XenofilteR package in R was used to filter out mouse-originated reads. Count tables and differential analysis were performed using EdgeR package in R.
Project description:Establishment of a heterozygous genome-wide loss-of-function CRISPR library for studying haploinsufficiency in human embryonic stem cells.
Project description:Interventions: Administraion of CPT-11 is twice for 4 weeks on days 1 and 15. CPT-11 is reconstituted in >=250 mL of normal saline or 5% dextrose in water and infuse 90min on day 1 for pharmacokinetics, and 90min over on day15. CPT-11 adjusted dosage is determined from 50,75,100,125 or 150mg/sqm in the heterozygous group and the homozygous group by continual reassessment method. CPT-11 dosage is fixed at 150mg/sqm in the wild group.Definision of UGT1A1 polymorphisms groups: The homozygous group is patient with homozygous genotype of UGT1A1*28/*28 or UGT1A1*6/*6, with combined heterozygous genotypes of UGT1A1*28 and UGT1A1*6. The heterozygous group is patient with heterozygous genotype of either UGT1A1*28 or UGT1A1*6. The wild group is patients with no UGT1A1*28 and UGT1A1*6 mutation.
Primary outcome(s): Maximum tolerated dose for the heterozygous group and the homozygous group, respectively. Incidence rate of dose limiting toxicities for the wild group.
Study Design: Single arm Non-randomized
Project description:Interventions: Patients receive FOLFIRI with bevacizumab fixed 5mg/kg. (Treatment will be continued unless the disease progression, unacceptable toxicity, or consent withdrawal.) Definision of UGT1A1 polymorphisms groups: The homozygous group is patient with homozygous genotype of UGT1A1*28/*28 or UGT1A1*6/*6, with combined heterozygous genotypes of UGT1A1*28 and UGT1A1*6. The heterozygous group is patient with heterozygous genotype of either UGT1A1*28 or UGT1A1*6. The wild group is patients with no UGT1A1*28 and UGT1A1*6 mutation. CPT-11 dosage is wild and heterozygous:CPT-11 150mg/m2 homozygous:CPT-11 100mg/m2
Primary outcome(s): 1)incidence of adverse events 2)frequency of severe toxicity
Study Design: Single arm Non-randomized
Project description:The transcription factor GATA2 plays a major role in the generation and maintenance of the hematopoietic system. In humans, heterozygous germline mutations in GATA2 often lead to a loss of function of one allele, causing GATA2 haploinsufficiency. In mice, Gata2 has an essential regulatory function in hematopoietic stem cell (HSC) generation and maintenance. However, whereas Gata2-null mice are lethal at embryonic day (E) 10.53, Gata2 heterozygous (Gata2+/-) mice survive to adulthood with normal blood values. However, mouse models thus emerged as a useful source to identify the function of GATA2 in HSC generation and fitness, they leave the mechanisms causing the different aspects of GATA2 deficiency syndrome largely undiscovered. Zebrafish have the advantage of having two GATA2 orthologues; Gata2a and Gata2b. Gata2a is expressed predominantly in the vasculature and is required for programming of the hemogenic endothelium. Gata2b is expressed in hematopoietic stem/progenitor cells (HSPCs) and homozygous deletion (gata2b-/-) redirects HSPCs differentiation bias, thus mimicking one of the GATA2 haploinsufficiency phenotypes found in patients. But patients carry heterozygous rather than homozygous GATA2 mutations, we specifically focused on how heterozygous Gata2b mutations could be mechanistically linked to erythro-myelodysplasia, a major clinical hallmark of GATA2 patients. To investigate the mechanisms of heterozygous GATA2 mutation caused GATA2 deficiency syndrome, we created a heterozygous gata2b mutation zebrafish model and sorted the entire progenitor and HSPC population including the lymphoid population from kidney marrow (KM) of WT and mutated zebrafish based on the scatter profile of flow cytometry for single-nucleus ATAC (snATAC) sequencing.
Project description:<p>We identified germline heterozygous mutations in <i>CTLA4</i> in members of four families with severe immune dysregulation. Human <i>CTLA4</i> haploinsufficiency caused dysregulation of FoxP3+ regulatory T (Treg) cells and lymphocytic infiltration of target organs, mimicking <i>Ctla4</i> homozygous mice. Patients also exhibited a B cell phenotype, with progressive loss of B cells and accumulation of autoreactive CD21<sup>lo</sup> B cells. This study demonstrates a critical quantitative role for CTLA-4 in human immune homeostasis.</p>
Project description:To clarify the effect of Mitf mutation on stria vascularis transcriptome and identify downstream-genes of Mitf pathway which responsible for hearing development, we employed high-throughput sequencing to analyze the transcriptome of MitfR/r and Mitfr/r stria vascularis. Compare the transcriptome difference between MITF mutated homozygous and heterozygous pigs.
Project description:The transcription factor GATA2 plays a major role in the generation and maintenance of the hematopoietic system. In humans, heterozygous germline mutations in GATA2 often lead to a loss of function of one allele, causing GATA2 haploinsufficiency. In mice, Gata2 has an essential regulatory function in hematopoietic stem cell (HSC) generation and maintenance. However, whereas Gata2-null mice are lethal at embryonic day (E) 10.53, Gata2 heterozygous (Gata2+/-) mice survive to adulthood with normal blood values. However, mouse models thus emerged as a useful source to identify the function of GATA2 in HSC generation and fitness, they leave the mechanisms causing the different aspects of GATA2 deficiency syndrome largely undiscovered. Zebrafish have the advantage of having two GATA2 orthologues; Gata2a and Gata2b. Gata2a is expressed predominantly in the vasculature and is required for programming of the hemogenic endothelium. Gata2b is expressed in hematopoietic stem/progenitor cells (HSPCs) and homozygous deletion (gata2b-/-) redirects HSPCs differentiation bias, thus mimicking one of the GATA2 haploinsufficiency phenotypes found in patients. But patients carry heterozygous rather than homozygous GATA2 mutations, we specifically focused on how heterozygous Gata2b mutations could be mechanistically linked to erythro-myelodysplasia, a major clinical hallmark of GATA2 patients. To investigate the mechanisms of heterozygous GATA2 mutation caused GATA2 deficiency syndrome, we created a heterozygous gata2b mutation zebrafish model and sorted the entire progenitor and HSPC population including the lymphoid population from kidney marrow (KM) of WT and mutated zebrafish based on the scatter profile of flow cytometry for single-cell RNA (scRNA) sequencing.