Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs
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ABSTRACT:
PROVIDER: PRJNA1091873 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28456505_1.fastq.gz | Fastqsanger.gz | |||
| SRR28456505_2.fastq.gz | Fastqsanger.gz | |||
| SRR28456506_1.fastq.gz | Fastqsanger.gz | |||
| SRR28456506_2.fastq.gz | Fastqsanger.gz | |||
| SRR28456509_1.fastq.gz | Fastqsanger.gz |
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Project description:Translating genetic findings for neurodevelopmental and psychiatric disorders (NPD) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, here we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop-codons (iSTOP) that lead to mRNA nonsense-mediated-decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 NPD genes. Using RNAseq, we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Interestingly, for three schizophrenia risk genes (SETD1A, TRIO, CUL1), despite the high efficiency of base editing, we only obtained heterozygous LoF alleles, suggesting their essential roles for cell growth. We replicated the reported neural phenotypes of SHANK3-haploinsufficiency and found CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.
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Project description:Scaled and Efficient Derivation of Loss of Function Alleles in Risk Genes for Neurodevelopmental and Psychiatric Disorders in Human iPSC [RNA-seq]
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Project description:Translating genetic findings for neurodevelopmental and psychiatric disorders (NPDs) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop codons (iSTOP) that lead to mRNA nonsense-mediated decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 genes. Using RNA sequencing (RNA-seq), we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Despite high editing efficiency, three schizophrenia risk genes (SETD1A, TRIO, and CUL1) only had heterozygous LoF alleles, suggesting their essential roles for cell growth. We found that CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.
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Project description:Translating genetic findings for neurodevelopmental and psychiatric disorders (NPD) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, here we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop-codons (iSTOP) that lead to mRNA nonsense-mediated-decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 NPD genes. Using RNAseq, we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Interestingly, for three schizophrenia risk genes (SETD1A, TRIO, CUL1), despite the high efficiency of base editing, we only obtained heterozygous LoF alleles, suggesting their essential roles for cell growth. We replicated the reported neural phenotypes of SHANK3-haploinsufficiency and found CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.
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Project description:Gender differences in the prevalence of psychiatric disorders are well documented in epidemiological studies. Exposure to stress during gestation impacts differentially between females and males. Environmental or biological factors can alter DNA methylation and affect the development of neurodevelopmental diseases. In this study, we explored gender-specific DNA methylation in regarding their potential connection to psychiatric outcomes. We assessed the methylation of cord blood of 39 females and 32 males born at term and with appropriate weight at birth. Mothers between 18 and 48 years of age (median: 28 years) were interviewed to gather information about environmental factors (gestational exposure) that can potentially interfere with the methylation profiles in the newborns. Bisulphite converted DNA was hybridized to Illumina HumanMethylation450 BeadChips. After exclusion of XYS probes, there were 2,332 differentially methylated CpG sites (DMS) between sexes, with an overlap of 890 (38%) CpG sites with an a cohort submitted to gestational stress exposition. Using externals datasets, we found that the DMSs were enriched within brain modules of co-methylated CpGs during gestation and were also differentially methylated in the brain comparing boys and girls during brain development. Genes associated to the DMSs were enriched for neurodevelopmental disorders. Accordingly, we described an enrichment of the DMSs among the differentially methylated CpG sites in brain tissue between schizophrenics and controls. Here, we suggest that gender changes methylation in the autosomes working as a primary driver to stress exposition which potentially contributes for gender differences found in psychiatric outcomes.
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