Need for high-resolution Genetic Analysis in iPSC: Results and Lessons from the ForIPS Consortium

Abstract

Genetic integrity of induced pluripotent stem cells (iPSCs) is essential for their validity as disease models and for potential therapeutic use. We describe the comprehensive analysis in the ForIPS consortium: an iPSC collection from donors with neurological diseases and healthy controls. Characterization included pluripotency confirmation, fingerprinting, conventional and molecular karyotyping in all lines. In the majority, somatic copy number variants (CNVs) were identified. A subset with available matched donor DNA was selected for comparative exome sequencing. We identified single nucleotide variants (SNVs) at different allelic frequencies in each clone with high variability in mutational load. Low frequencies of variants in parental fibroblasts highlight the importance of germline samples. Somatic variant number was independent from reprogramming, cell type and passage. Comparison with disease genes and prediction scores suggest biological relevance for some variants. We show that high-throughput sequencing has value beyond SNV detection and the requirement to individually evaluate each clone.

TRIM28 haploinsufficiency predisposes to Wilms tumor

“…Enrichment and library preparation for exome sequencing on individuals 3, 4, 7 and 8 was performed using the SureSelect Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA), and sequencing was carried out with 125 bp paired‐end reads on an Illumina Hiseq 2,500 system (Illumina, Inc., San Diego, CA). Concurrent somatic variant calling using freebayes v1.1.0 was performed as described previously on the final 8 tumor and 6 germline BAM files from the affected individuals, and 40 in‐house control samples . Details on variant calling are available in Supporting Information methods.…”

“…Concurrent somatic variant calling using freebayes v1.1.0 26 was performed as described previously on the final 8 tumor and 6 germline BAM files from the affected individuals, and 40 in-house control samples. 27 Details on variant calling are available in Supporting Information methods.…”

Stem cell derived oligodendrocytes to study myelin diseases

“…Therefore, transplantation of iPSC derived NSC or oligodendroglial lineage cells represent an attractive alternative although the potential immunogenicity even of autologous iPSC has been debated (Araki et al, ; Zhao, Zhang, Rong, & Xu, ). One challenge regarding the iPSC technology is the genetic instability associated with reprogramming and the risk to accumulate mutations due to their high proliferation rate; for example approximately 70% of iPSC cell lines display copy number variations (Popp et al, ). This represents a significant caveat for transplantation experiments and resulted in a hold of the first clinical trial in which autologous retinal pigment epithelial (RPE) for treatment of macular degeneration were transplanted (Mandai et al, ).…”

A biallelic truncating AEBP1 variant causes connective tissue disorder in two siblings

“…The clinical exome BAM file was concurrently called with 44 in‐house controls using the UnifiedGenotyper (DePristo et al, ) from GATK version 3.8–1‐0‐gf15c1c3ef to produce a multisample VCF file. Genotypes at SNP positions were extracted from the CMA of individual D:II‐1 as described previously and joined into multisample VCF file with 23 controls from a recent project (Popp et al, ). The het command from PLINK software (Purcell et al, ) v1.90b4 was used on both these VCF files to compute the inbreeding coefficient F.…”