High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs

Abstract

The KOLF2.1J iPSC line was recently proposed as a reference iPSC to promote the standardization of research studies in the stem cell field. Due to overall good performance differentiating to neural cell lineages, high gene editing efficiency, and absence of genetic variants associated to neurological disorders KOLF2.1J iPSC line was particularly recommended for neurodegenerative disease modeling. However, our work uncovers that KOLF2.1J hPSCs carry heterozygous small copy number variants (CNVs) that cause DTNBP1, JARID2 and ASTN2 haploinsufficiencies, all of which are associated with neurological disorders. We further determine that these CNVs arose in vitro over the course of KOLF2.1J iPSC generation from a healthy donor-derived KOLF2 iPSC line and affect the expression of DNTBP1, JARID2 and ASTN2 proteins in KOLF2.1J iPSCs and neural progenitors. Therefore, our study suggests that KOLF2.1J iPSCs carry genetic variants that may be deleterious for neural cell lineages. This data is essential for a careful interpretation of neural cell studies derived from KOLF2.1J iPSCs and highlights the need for a catalogue of iPSC lines that includes a comprehensive genome characterization analysis.

Figure 1:. High density SNP array uncovers CNVs affecting coding genes in Chr6p22 and Chr9q33 in KOLF2.1J iPSCs.

(A) Chromosome 6 cytoband schematics (top) and Log R Ratio (LRR) and B Allele frequency (BAF) plots (bottom) show reduction of signal intensity and a loss of heterozygosity in 6p22 region of unedited and p2 KOLF2.1J iPSCs compared to a control iPSC line. (B) Chromosome 9 cytoband schematics (top) and LRR and BAF plots (bottom) show reduction of signal intensity and a loss of heterozygosity in 9q33 region of KOLF2.1J iPSCs unedited and at p2 compared to a control iPSC line.

Figure 2:. gDNA qPCR analysis confirms the presence of chr6p22 and chr9q33 CNV in KOLF2.1J iPSCs.

(A) Schematic representation of Chromosome 6 (Chr6:15,200,000–15,900,000). Window illustrates the 0.2 Mb deleted region (red bar) and affected genes, JARID2 (green bar) and DTBNP1 (blue bar). (B-I) Genomic DNA qPCR results showing half levels of amplification within the Chr6 deleted CNV region in KOLF2.1J compared to control iPSCs (D-G) and regions upstream and downstream of the deletion (A-B and H-I). (J) Schematic representation of Chromosome 9 (Chr9:116,200,000–119,400,000) with the 0.1 Mb deleted region (red bar) and affected genes, ASTN2 (purple bar) and ASTN-AS1 (yellow bar). (K-P) Genomic DNA qPCR results showing half levels of amplification within the Chr9 deleted CNV region in KOLF2.1J compared to control iPSCs (L-N) and regions upstream and downstream of the deletion (K and P). Graphs show mean +/−SD of n=3 control hPSC lines and n=4 KOLF2.1J iPSC independent stocks. p-values were calculated with a two-sided unpaired t-test. ns=non-significant. Grey filled arrowheads indicate exons. > and < symbols indicate qPCR primer pair positions.

Figure 3:. Genome sequencing reanalysis confirms CNVs at Chr6p22 and Chr9q33.

(A, B) Log2 ratio and BAF plots show reduction of signal intensity (Log2 ratio) and a loss of heterozygosity (BAF) in Chr6p22 (A) and Chr9q33 (B) CNV regions compared to diploid flanking regions. Shadowed area represents the CNV defined by the SNP array and vertical lines the base-resolution breakpoints determined from the genome sequencing data. Chromosome 6 and 9 cytoband schematics with the CNV breakpoints at base resolution are shown on the top. (C, D) Sanger sequencing of PCR amplicons including ~200bp up- and downstream of the predicted breakpoints confirm deletion CNVs in Chr6(C) and Chr9 (D) and adjust the breakpoint positions. Nucleotides shadowed in grey (D) are part of the 56 bp long microhomology domain of two AluS repetitive elements localized in Chr9 CNV breakpoints.

Figure 4:. Chr6p22 and Chr9q33 CNVs arose in vitro and affect the expression of JARID2, DTNBP1 and ASTN2 genes.

(A, B) LRR and BAF plots of the KOLF2 iPSC line SNP array show diploid Chr6 (A) and Chr9 (B) regions, indicating that the CNVs arose in culture between KOLF2 to KOLF2.1J generation. Horizontal lines and red dots depict SNPs that fall within KOLF2.1J CNV regions. (C, D) Quantitative proteomic data retrieved from Nam et al show that JARID2, DTNBP1 and ASTN2 protein levels are ~half in KOLF2.1J compared to the reference H9 line at both pluripotency (C) and neural progenitor stage (D). Graphs show mean +/−SD of n=4 cultures and q-values as calculated in the reference proteomic analysis with Welsch’s t-test and FDR correction for multiple comparisons.