Divergent Levels of Marker Chromosomes in an hiPSC-Based Model of Psychosis.

paper Cited Public

Authors: Tcw, Julia; Carvalho, Claudia M B; Yuan, Bo; Gu, Shen; Altheimer, Alyssa N; McCarthy, Shane; Malhotra, Dheeraj; Sebat, Jonathan; Siegel, Arthur J; Rudolph, Uwe; Lupski, James R; Levy, Deborah L; Brennand, Kristen J
Year: 2017
Journal: Stem cell reports
PMID: 28216146
DOI: 10.1016/j.stemcr.2017.01.010
PMCID: PMC5355568

Figure 1

Identification of 9p24.1 DUP/TRP and a Mar Chromosome in Two Patients with Psychotic Disorders(A) Schematic of human chr 9, highlighting band 9p24.1, which contains the DUP/TRP region. Encoded genes are listed on the right; genes in the DUP region (red) and TRP region (blue) are indicated (GLDC is in the TRP region).(B) From leukocytes, custom aCGH identified a CGR in the proband DL3363 (shown) and mother DL5459. This structural rearrangement, constituted by a DUP/TRP, spans 1.85 Mb.(C) List of HF lines used in this study.(D and E) Karyotype analysis of HF lines derived from the 9p24.1-carrier proband (DL3363) and mother (DL5459). Representative karyotype images of mar (M) (D) and percentage mosaicism (E) of HF lines derived from the 9p24.1-carrier proband (DL3363) and mother (DL5459). Red arrows indicate an additional t(1;18)(p22;q11.2) abnormality identified in 3/20 fibroblast cells (but not present in validated hiPSC lines).(F and G) GLDC FISH analysis of HF lines derived from the 9p24.1-carrier proband (DL3363) and mother (DL5459). FISH images (600× magnification, images cropped and resized post capture) (F) from HFs from the proband (DL3363) (shown) and his mother (DL5459) with two (top), three (middle), and four (bottom) GLDC probes (red) within the DAPI-positive nucleus. Proportion of HF cells (G) with GLDC FISH probes in early passage (p5) and late passage (p20) from DL3363 and DL5459 (number of cells = 200 each, one assay per line).

Figure 2

Serendipitous Generation of 9p24.1-Positive and 9p24.1-Negative hiPSCs from Two 9p24.1-Carrier Patients(A and B) Validation of hiPSCs derived from the 9p24.1-carrier proband (DL3363) and mother (DL5459). Immunofluorescence for NANOG, OCT4, TRA1-81, and TRA-1-60 (A) and fluorescence-activated cell sorting histograms for TRA-1-60 and SSEA4 (B) from hiPSCs derived from the 9p24.1-carrier proband (DL3363) and mother (DL5459). Scale bar, 200 μm. Representative images per condition are selected from Figure S2A.(C) Representative Agilent aCGH plots of the 9p24.1 region reveals the presence of the DUP/TRP rearrangement in some hiPSCs (9p24.1-positive) generated from the proband but not in others (9p24.1-negative isogenic hiPSCs).(D and E) Karyotype analysis of hiPSCs derived from the 9p24.1-carrier proband (DL3363) and mother (DL5459). Representative karyotype images of mar (M) (D) and percentage mosaicism (E) of hiPSCs derived from the 9p24.1-carrier proband (DL3363) and mother (DL5459). Karyotype counts indicated variable levels of mosaicism of the mar element in all genotype-positive hiPSCs.(F) Bright-field images of high mar (60%) and low mar (10%) 9p24.1-carrier hiPSCs. Scale bar, 200 μm.

Figure 3

In 9p24.1-Carrier hiPSCs, the Mar Contains Sequence of Chr 9 and the Triplicated Gene GLDC(A–C) Chr 9 FISH analysis of hiPSCs from the 9p24.1-carrier proband (DL3363) and mother (DL5459). FISH images (600× magnification, images cropped and resized post capture) (A) with two (left) or three (right) chr 9 probes (red) within the DAPI-positive nucleus as well as approximate correlation (B) between karyotypic mar mosaicism and chr 9 FISH mosaicism. FISH image (600× magnification, image cropped and resized post capture) (C) from a 9p24.1-carrier hiPSC showing overlap of the chr 9 probe with the mar element (arrow).(D–F) GLDC FISH analysis of hiPSCs derived from the 9p24.1-carrier proband (DL3363) and mother (DL5459). FISH images (600× magnification, images cropped and resized post capture) (D) and quantitative analysis (number of cells = 200 each, one assay per line) (E) from high mar and low mar of 9p24.1-carrier (C1–C3) and non-carrier (NC1–NC3) hiPSCs at low (p12) and high (p24) passage with three (left) and four (right) GLDC probes (red) within the DAPI-positive nucleus. FISH image (600× magnification, image cropped and resized post capture) (F) from 9p24.1-carrier hiPSCs showing overlap of the GLDC probe with the mar element (arrow). C, carrier; NC, non-carrier.(G) qRT-PCR for GLDC mRNA levels in HFs as well as carrier and non-carrier hiPSCs from the 9p24.1-carrier proband (DL3363) and mother (DL5459). Percentages of mar in each cell line are indicated. Data are shown as mean ± SD from three technical replicates of each individual line. One-way ANOVA with Tukey's multiple comparison test: n.s., not significant; ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

Neural Differentiation of 9p24.1 High and Low Mar hiPSCs Yields Similar Levels of Mar Elements(A and B) Bright-field (A) and immunofluorescence for FOXP2, NESTIN, and PAX6 (B) images of NPCs from high (55.0%) and low mar (10.0%) 9p24.1-carrier hiPSCs (DL5459 C5 and C6, respectively). Scale bars, 500 μm.(C and D) FISH images (600× magnification, images cropped and resized post capture) (C) and quantitative analysis (number of cells = 200 each, one assay per line) (D) from NPCs differentiated from high (55.0%) and low mar (10.0%) 9p24.1-carrier hiPSCs (DL5459 C5 and DL5459 C6) with three (left) and four (right) GLDC probes (red) within the DAPI-positive nucleus.

#	Section	Preview
0	Introduction	Chromosomal trisomy disorders have long been associated with abnormal developmental outcomes…
1	Introduction	Growing evidence suggests that hiPSCs are fundamentally similar regardless of reprogramming methods…
2	Introduction	process (Ma et al., 2014). Genetic and epigenetic errors can distinguish hiPSC lines from the same…
3	Introduction	We present evidence that although extrachromosomal marker (mar) elements are relatively stable…
4	Results — Identification of a Duplication/Triplication of 9p24.1 in Two Patients with Psychotic Disorders	A small family cohort was identified in which a CGR involving chr 9p24.1 occurred in two carriers,…
5	Results — The 9p24.1 DUP/TRP Exists on a Marker Chromosome	Human fibroblasts (HFs) were obtained from the two 9p24.1 DUP/TRP carriers; unexpectedly, G-band…
6	Results — The 9p24.1 DUP/TRP Exists on a Marker Chromosome	Mar elements are often believed to have low stability, as they can be lost during cell division due…
7	Results — Reprogramming of 9p24.1-Carrier HFs Yields Isogenic Carrier and Non-carrier hiPSCs; 9p24.1-Carrier hiPSCs Show Variable Mosaicism in All Lines	We generated genetically unmanipulated and presumably clonal hiPSCs from the two 9p24.1 carriers…
8	Results — Reprogramming of 9p24.1-Carrier HFs Yields Isogenic Carrier and Non-carrier hiPSCs; 9p24.1-Carrier hiPSCs Show Variable Mosaicism in All Lines	We analyzed hiPSCs from both the proband (DL3363) and mother (DL5459) for the percentage presence of…
9	Results — Mar Element Contains Sequence of Chr 9 and the Triplicated Gene GLDC	FISH in interphase cells confirmed that 9p24.1-carrier hiPSCs from both the proband (DL3363) and…
10	Results — Mar Element Contains Sequence of Chr 9 and the Triplicated Gene GLDC	When we compared the frequency of mosaicism in high and low mar hiPSCs by GLDC FISH at p12 (DL5459…
11	Results — Mar Element Contains Sequence of Chr 9 and the Triplicated Gene GLDC	Perhaps reflecting the low expression of GLDC in HFs relative to NPCs, qPCR for GLDC expression…
12	Results — Neural Differentiation of 9p24.1 High and Low Mar hiPSCs Can Yield NPCs with Similar Levels of Mar Elements	We tested the effect of neural differentiation on the frequency of mar mosaicism. When we compared…
13	Discussion	Mar elements are rare in the general population (estimated to occur in 0.044% of newborn infants)…
14	Discussion	As future hiPSC-based studies include a growing number of patients with rare CGRs, they are likely…
15	Discussion	Instability of chromosomal abnormalities has been well documented in vivo and in vitro. Among…
16	Discussion	The presence of mar mosaicism in source HFs, presumably clonal hiPSCs and hiPSC NPCs, indicates that…
17	Discussion	pair with some sensitive synaptic staining protocols, many commercial MEA plates are not amenable to…
18	Discussion	The reversion of the mutation to wild-type may reflect a selection disadvantage to 9p24.1 cells in…
19	Experimental Procedures — Patients, hiPSC Derivation, and NPC Differentiation	Human subjects work was approved by the McLean Hospital Institutional Review Board. HFs were…

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In this knowledge base

Title	Year	PMID
Genetics of Alcohol Use Disorder: A Role for Induced Pluripotent Stem Cells?	2018	29897633

External

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An increased copy number of glycine decarboxylase (GLDC) associated with psychosis reduces extracellular glycine and impairs NMDA receptor function.	Kambali M et al.	—	2025	→
Glycine Reverses Behavioral Deficits in a Mouse Model for Psychosis With 4 Copies of the Gldc Gene.	Wang M et al.	—	2025	→
Upregulated GIRK2 Counteracts Ethanol-Induced Changes in Excitability and Respiration in Human Neurons.	Prytkova I et al.	—	2024	→
A marker chromosome in psychosis identifies glycine decarboxylase (GLDC) as a novel regulator of neuronal and synaptic function in the hippocampus	Kambali M et al.	—	2023	—
Investigation of Schizophrenia with Human Induced Pluripotent Stem Cells.	Powell SK et al.	—	2020	→
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Genetics of Alcohol Use Disorder: A Role for Induced Pluripotent Stem Cells?	Prytkova I et al.	—	2018	→
Marker chromosome genomic structure and temporal origin implicate a chromoanasynthesis event in a family with pleiotropic psychiatric phenotypes.	Grochowski CM et al.	—	2018	→
Personalized medicine in a dish: the growing possibility of neuropsychiatric disease drug discovery tailored to patient genetic variants using stem cells.	Brennand KJ	—	2017	→