Functional Dissection of Chromosome 7q Loss and Haploinsufficient Gene Discovery Using iPSC Models of MDS
Authors: Kotini Andriana , Delrow Jeffrey J , Graubert Timothy A. , Nimer Stephen , Papapetrou Eirini P 
Published: October 2019

Abstract
Somatic loss of one copy of the long arm of chromosome 7 [del(7q)] is a characteristic cytogenetic abnormality in MDS and other myeloid malignancies, well-recognized for decades and associated with unfavorable prognosis. Despite compelling clinical evidence that the del(7q) holds a key to the pathogenesis of MDS, the mechanism remains elusive. Gene haploinsufficiency has been proposed as a plausible mechanism, but definitive evidence is lacking. Narrowing down the responsible region and identifying the critical genes has proved challenging with existing approaches. Chr7q deletions are typically very large and modeling in the mouse is problematic, as the genomic regions syntenic to the human chr7q are dispersed into 4 different mouse chromosomes. More than one commonly deleted regions (CDRs) have been proposed by physical mapping studies in patient cells. A handful of genes on chr7q have been implicated through candidate gene approaches and knockout studies in the mouse. However, despite the intense efforts, the contribution of the del(7q) to the disease phenotype and the critical gene or genes on chr7q that mediate it remain unclear.
To overcome the limitations of existing tools (primary patient cells, mouse models) to study del(7q)-MDS, we developed a new model harnessing reprogramming and genome editing technologies. First we derived del(7q)-, in parallel with isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from bone marrow hematopoietic cells of two MDS patients. By whole exome sequencing, we were able to identify somatic variants of the MDS clone and show that they are present in the del(7q)-MDS-iPSCs, but not in the karyotypically normal iPSCs, which therefore unambiguously originate from residual normal cells. We used these isogenic and fully genetically characterized patient-derived iPSCs to characterize disease-relevant cellular phenotypes specific to the MDS-iPSCs, which included severely reduced hematopoietic potential and clonogenicity and increased apoptosis.
We next found that iPSC clones spontaneously acquiring a second copy of chr7q had an in vitro growth advantage, which enabled us to isolate one clone that completely rescued its hematopoietic differentiation ability upon restoration of a diploid dosage of a ~30Mb chr7q telomeric region. This result provides the first definitive evidence that the del(7q) abnormality confers a profound loss of hematopoietic potential and that this defect is mediated through reduced dosage, consistent with haploinsufficiency of one or more genes.
To further narrow down the critical region, we developed genome editing technologies to engineer large chromosomal deletions for the first time in human cells. Combining gene targeting with a modified Cre-loxP approach and the CRISPR/Cas9 endonuclease technology, we were able to generate a panel of 12 iPSC lines harboring hemizygous deletions of various defined segments spanning the entire long arm of chr7. By asking which of them recapitulate the MDS hematopoietic phenotype, we were able to “functionally map” the critical segment in a region spanning cytobands q32.3 - q36.1.
To identify critical gene(s) on chr7q, we designed a phenotype-rescue screen. We selected 62 candidate haploinsufficient genes on the basis of significantly reduced expression in del(7q)- compared to isogenic normal iPSCs. We constructed a barcoded lentiviral library of these ORFs and performed a pooled library screen for rescue of hematopoiesis in del(7q)-MDS-iPSCs, i.e. enrichment in CD45+ hematopoietic progenitors. We selected the top 6 genes within our region that were found recurrently enriched in at least 2 independent experiments. Four of them could be individually validated: dosage complementation partially rescued hematopoiesis and knockdown studies mimicking haploinsufficiency (50% knockdown) in normal primary CD34+ hematopoietic progenitor cells had a detrimental effect in hematopoiesis. The four genes include EZH2 and LUC7L2 – two genes found to harbor recurrent heterozygous loss-of-function mutations in MDS – as well as two genes with no previously known role in MDS, located in close genomic proximity to the former two.
This approach, constituting a new paradigm of functional human genetics with patient-specific iPSCs, can be more broadly applicable to the study of the phenotypic consequences of segmental chromosomal deletions and to haploinsufficient gene discovery.

Disclosures
No relevant conflicts of interest to declare.