Biology subjects

Chien, W.-M.

Publications and source records attributed to Chien, W.-M..

5 recordsLinked to original sources

bioRxiv2023

Multiplexed functional assessments of MYH7 variants in human cardiomyocytes at scale

BackgroundSingle, autosomal-dominant missense mutations in MYH7, which encodes a sarcomeric protein (MHC-{beta}) in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically-actionable. However, [~]75% of MYH7 variants are of unknown significance (VUS), causing diagnostic challenges for clinicians and emotional distress for patients. Deep mutational scans (DMS) can determine variant effect at scale, but have only been utilized in easily-editable cell lines. While human induced pluripotent stem cells (hiPSCs) can be differentiated to numerous cell types that enable the interrogation of variant effect in a disease-relevant context, DMS have not been executed using diploid hiPSC derivates. However, CRaTER enrichment has recently enabled the pooled generation of a saturated five position MYH7 variant hiPSC library suitable for DMS for the first time. ResultsAs a proof-of-concept, we differentiated this MYH7 variant hiPSC library to cardiomyocytes (hiPSC-CMs) for multiplexed assessment of MHC-{beta} variant abundance by massively parallel sequencing (VAMP-seq) and hiPSC-CM survival. We confirm MHC-{beta} protein loss occurs in a failing human heart with a pathogenic MYH7 mutation. We find the multiplexed assessment of MHC-{beta} abundance and hiPSC-CM survival both accurately segregate all pathogenic variants from synonymous controls. Overall, functional scores of 68 amino acid substitutions across these independent assays are [~]50% consistent. ConclusionsThis study leverages hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants at scale for the first time. This proof-of-concept demonstrates the ability to DMS previously restricted, clinically-actionable genes to reduce the burden of VUS on patients and clinicians.

genetics↗

bioRxiv2023

CRaTER enrichment for on-target gene-editing enables generation of variant libraries in hiPSCs

Standard transgenic cell line generation requires screening 100-1000s of colonies to isolate correctly edited cells. We describe CRISPRa On-Target Editing Retrieval (CRaTER) which enriches for cells with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of single nucleotide variants (SNVs) in MYH7, a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different MYH7 SNVs. We differentiated these hiPSCs to cardiomyocytes and show MYH7 fusion proteins can localize as expected. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of transgenic cell lines at unprecedented scale.

genetics↗

bioRxiv2023

Cardiomyocyte apoptosis contributes to contractile dysfunction in stem cell model of MYH7 E848G hypertrophic cardiomyopa-thy

Missense mutations in myosin heavy chain 7 (MYH7) are a common cause of hyper-trophic cardiomyopathy (HCM), but the molecular mechanisms underlying MYH7-based HCM remain unclear. In this work, we generated cardiomyocytes derived from isogenic human induced pluripotent stem cells to model the heterozygous pathogenic MYH7 missense variant, E848G, which is associated with left ventricular hypertrophy and adultonset systolic dysfunction. MYH7E848G/+ increased cardiomyocyte size and reduced the maximum twitch forces of engineered heart tissue, consistent with the systolic dysfunction in MYH7 E848G HCM patients. Interestingly, MYH7E848G/+ cardiomyocytes more frequently underwent apoptosis that was associated with increased p53 activity relative to controls. However, genetic ablation of TP53 did not rescue cardiomyocyte survival or restore engineered heart tissue twitch force, indicating MYH7E848G/+ cardiomyocyte apoptosis and contractile dysfunction are p53-independent. Overall, our findings suggest that cardiomyocyte apoptosis plays an important role in the MYH7E848G/+ HCM phenotype in vitro and that future efforts to target p53-independent cell death pathways may be beneficial for the treatment of HCM patients with systolic dysfunction.

molecular biology↗

bioRxiv2020

Novel TREM2 splicing isoform that lacks the V-set immunoglobulin domain is abundant in the human brain

TREM2 is an immunoglobulin-like receptor expressed by certain myeloid cells, such as macrophages, dendritic cells, osteoclasts and microglia. In the brain, TREM2 plays an important role in the immune function of microglia, and its dysfunction is linked to various neurodegenerative conditions in humans. Ablation of TREM2 or its adaptor protein TYROBP causes Polycystic Lipo-Membranous Osteodysplasia with Sclerosing Leukoencephalopathy (also known as Nasu-Hakola disorder) with early onset of dementia, while some missense variants in TREM2 are associated with an increased risk of late-onset Alzheimers disease. Human TREM2 gene is a subject to alternative splicing, and its major, full-length canonical transcript encompasses 5 exons. Herein, we report a novel alternatively spliced TREM2 isoform without exon 2 ({Delta}e2), which constitutes a sizable fraction of TREM2 transcripts and has highly variable inter-individual expression in the human brain (average frequency 10%; range 3.7-35%). The protein encoded by {Delta}e2 lacks a V-set immunoglobulin domain from its extracellular part but retains its transmembrane and cytoplasmic domains. We demonstrated {Delta}e2 protein expression in TREM2-positive THP-1 cells, in which the expression of full-length transcript was precluded by CRISPR/Cas9 disruption of the exon 2 coding frame. In "add-back" experiments, overexpression of full-length, but not {Delta}e2 TREM2, restored phagocytic capacity and promoted interferon type I response in the knockout cells. Our findings suggest that expression of a {Delta}e2 splice isoform may modify the dosage of full-length transcript potentially weakening some TREM2 receptor functions in the human brain.

immunology↗

bioRxiv2018

TREM2 R47H exacerbates immune response in Alzheimer’s disease brain

The R47H variant in the microglial TREM2 receptor is a strong risk factor for Alzheimers disease (AD). To characterize processes affected by R47H we performed integrative network analysis of genes expressed in brains of AD patients with R47H, sporadic AD without the variant and patients with polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), a systemic disease with early onset dementia caused by loss-of function mutations in TREM2 or its adaptor TYROBP. While sporadic AD had few perturbed microglial and immune genes, TREM2 R47H AD demonstrated upregulation of interferon type I response and pro-inflammatory cytokines accompanied by induction of NKG2D stress ligands. In contrast, PLOSL had distinct sets of highly perturbed immune and microglial genes that included inflammatory mediators, immune signaling, cell adhesion and phagocytosis. TREM2 knock-out in THP1, a human myeloid cell line that constitutively expresses the TREM2-TYROBP receptor, inhibited response to the viral RNA mimetic poly(I:C), and overexpression of ectopic TREM2 restored the response. Compared to wild type protein, R47H TREM2 had higher stimulatory effect on the interferon type I response signature. Our findings point to a role of the TREM2 receptor in the control of the interferon type I response in myeloid cells and provide insight regarding the contribution of R47H TREM2 to AD pathology.

genetics↗