Biology subjects

Kwan, V.

Publications and source records attributed to Kwan, V..

3 recordsLinked to original sources

bioRxiv2018

CNTN5-/+ or EHMT2-/+ iPSC-Derived Neurons from Individuals with Autism Develop Hyperactive Neuronal Networks

Induced pluripotent stem cell (iPSC)-derived cortical neurons are increasingly used as a model to study developmental aspects of Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of rare (penetrant) variant(s) and common (weaker) polygenic risk variant(s) to ASD, \"isogenic\" iPSC-derived neurons from probands and family-based controls, for modeling, is critical. We developed a standardized set of procedures, designed to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD (14 ASD-affected individuals, 3 unaffected siblings, 8 unaffected parents). Heterozygous de novo (7 families; 16p11.2, NRXN1, DLGAP2, CAPRIN1, VIP, ANOS1, THRA) and rare-inherited (2 families; CNTN5, AGBL4) presumed-damaging variants were characterized in ASD risk genes/loci. In three additional families, functional candidates for ASD (SET), and combinations of putative etiologic variants (GLI3/KIF21A and EHMT2/UBE2I combinations in separate families), were modeled. We used a large-scale multi-electrode array (MEA) as our primary high-throughput phenotyping assay, followed by patch clamp recordings. Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. Our biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research.

neuroscience↗

bioRxiv2018

Complete Disruption of Autism-Susceptibility Genes by Gene-Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons

Autism Spectrum Disorder is phenotypically and genetically heterogeneous, but genomic analyses have identified candidate susceptibility genes. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of 10 ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NEUROG2)-directed differentiation of iPSCs allowed production of cortical excitatory neurons, and mutant proteins were not detectable. RNAseq revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory post-synaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2- and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.

neuroscience↗

bioRxiv2018

A Patient-Derived Cellular Model for Huntington Disease Reveals Phenotypes at Clinically Relevant CAG Lengths

The huntingtin protein participates in several cellular processes that are disrupted when the polyglutamine tract is expanded beyond a threshold of 37 CAG DNA repeats in Huntingtons disease (HD). Cellular biology approaches to understand these functional disruptions in HD have primarily focused on cell lines with synthetically long CAG length alleles that clinically represent outliers in this disease and a more severe form of HD that lacks age-onset. Patient-derived fibroblasts are limited to a finite number of passages before succumbing to cellular senescence. We used human telomerase reverse transcriptase (hTERT) to immortalize fibroblasts taken from individuals of varying age, sex, disease onset and CAG repeat length, which we have termed TruHD cells. TruHD cells display classic HD phenotypes of altered morphology, size and growth rate, increased sensitivity to oxidative stress, aberrant ADP/ATP ratios and hypophosphorylated huntingtin protein. We additionally observed dysregulated ROS-dependent huntingtin localization to nuclear speckles in HD cells. We report the generation and characterization of a human, clinically relevant cellular model for investigating disease mechanisms in HD at the single cell level, which, unlike transformed cell lines, maintains TP53 function critical for huntingtin transcriptional regulation and genomic integrity.

cell biology↗