Using CRISPR/Cas9 genome editing in human iPSCs for deciphering the pathogenicity of a novel CCM1 transcription start site deletion

Abstract

Cerebral cavernous malformations are clusters of aberrant vessels that can lead to severe neurological complications. Pathogenic loss-of-function variants in the CCM1, CCM2, or CCM3 gene are associated with the autosomal dominant form of the disease. While interpretation of variants in protein-coding regions of the genes is relatively straightforward, functional analyses are often required to evaluate the impact of non-coding variants. Because of multiple alternatively spliced transcripts and different transcription start points, interpretation of variants in the 5′ untranslated and upstream regions of CCM1 is particularly challenging. Here, we identified a novel deletion of the non-coding exon 1 of CCM1 in a proband with multiple CCMs which was initially classified as a variant of unknown clinical significance. Using CRISPR/Cas9 genome editing in human iPSCs, we show that the deletion leads to loss of CCM1 protein and deregulation of KLF2, THBS1, NOS3, and HEY2 expression in iPSC-derived endothelial cells. Based on these results, the variant could be reclassified as likely pathogenic. Taken together, variants in regulatory regions need to be considered in genetic CCM analyses. Our study also demonstrates that modeling variants of unknown clinical significance in an iPSC-based system can help to come to a final diagnosis.

Endothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature

“…We have recently established a novel CCM1 knockout iPSC model and demonstrated that CCM1 −/− iPSCs can be differentiated into ECs which show the upregulation of KLF2 and KLF4 as well as downregulation of THBS1 [ 20 ]. These gene expression differences are well-characterized consequences of CCM1 inactivation in ECs [ 9 , 21 ].…”

“…The CCM1 +/+ and CCM1 −/− iPSC lines used in this study were derived from the parental AICS-0023 iPSC line (Allen Cell Collection, Coriell Institute, Camden, NJ, USA) before [ 20 ]. IPSCs were maintained in Essential 8 Flex medium (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO 2 on plates coated with growth factor reduced Matrigel (Corning, New York, NY, USA).…”

“…The CRISPR/Cas9 genome editing protocol to generate the CCM1 −/− iPSC lines used in this study was already described before [ 20 ]. In brief, AICS-0023 iPSCs were transfected with single guide RNA (sgRNA):Cas9 ribonucleoprotein complexes with the target region located in exon 10 of CCM1 (LRG_650t1, 5′-GGAGCTCCTAGACCAAAGTA-3′; Integrated DNA Technologies, Coralville, IA, USA) using Lipofectamine Stem Transfection Reagent (Thermo Fisher Scientific).…”

“…Furthermore, non-genetic third hits are believed to play a critical role in CCM genesis [ 19 ]. To expand our knowledge on the role of CCM1 in non-endothelial cell types and the importance of exogenous triggers in CCM formation, we combined our recently established isogenic CCM1 knockout induced pluripotent stem cell ( CCM1 −/− iPSC) model [ 20 ], the differentiation of iPSCs into endothelial-like cells (hereafter referred to as ECs), and RNA sequencing analyses. We demonstrate that the loss of CCM1 expression leads to strong gene expression differences in ECs but not in iPSCs or early mesoderm progenitor cells (eMPCs).…”

Cas9-Mediated Nanopore Sequencing Enables Precise Characterization of Structural Variants in CCM Genes

“…For example, they can hardly identify the precise breakpoints of CNVs, pathogenic variants in non-coding regions, and complex structural variants (SVs), e.g., inversions or interchromosomal insertions. This is particularly relevant since all these types of genetic variation have been described for CCM patients in the recent literature [ 6 , 7 , 8 , 9 ]. In the context of analyses for CNVs segregating within CCM families, cost-effectiveness is also an issue.…”