Balancing disease and productivity: recapitulating a Bardet-Biedl syndrome associated livestock mutation in pig induced pluripotent stem cells

The selective breeding of livestock for desirable phenotypic attributes has revealed cases where mutant heterozygotes display an advantageous phenotype despite detrimental homozygous traits. This is a form of balancing selection known as heterozygote advantage. A 212kb deletion in pigs causes increased food intake and growth of heterozygotes, whilst homozygotes die during embryonic development. The deletion truncates BBS9 (a ubiquitous but lowly expressed protein that acts as part of the BBSome complex involved in primary cilia homeostasis) while deleting two putative enhancers for the downstream gene BMPER. BMPER is a secreted glycoprotein which modulates BMP signalling. This project aimed to investigate the cause of the observed phenotypes using pig induced pluripotent stem cells (iPSCs) as an in vitro model. The goals of this research were to 1) assess the likelihood of BMPER expression being impacted by the deletion, 2) investigate the expression of BBS9 in the developing embryo, 3) create BBS9 mutant pig iPSC lines, and 4) characterise mutants to assess the functionality of the truncated BBS9 protein. Literature research indicated the presence of several enhancer regions within the deletion locus, some of which are known BMPER enhancers. Therefore, it is likely that the mutation impacts BMPER expression. The chicken embryo, being a model system for studying embryonic patterns of gene expression and its accessibility, was utilised in HCR RNA-FISH experiments to study BBS9 expression. Ubiquitous low-level expression of BBS9 was present throughout the embryo, with elevated expression in the developing retina and floor plate of the neural tube. Multiple isoforms of BBS9 were shown to exist in both pigs and chickens. Frameshift knock-in cell lines (BBS9694-RFP) that recapitulate the truncated BBS9 protein observed in the 212kb deletion, and two BBS9 knockout cell lines were created. From sequencing, BBS9694-RFP, all were either heterozygous or had mutations that did not cause a frameshift but had unknown impacts on the other allele. Characterisation of all BBS9 mutants was genomic, as protein characterisation could not be determined due to antibody incompatibility. Immunofluorescence for ciliary markers showed a significant reduction in ciliary expression of the BBS9 KO lines compared to WT parental and subclonal iPSC lines; two of the truncated BBS9 lines also showed decreased ciliary expression as determined by the percentage of ciliated centrosomes; the other truncated lines had no change in ciliary expression but did display morphological and growth differences. The ability of the BBS9 mutants to respond to sonic hedgehog (SHH) signalling – a well-described ciliary-dependent signalling pathway - was assessed via qPCR for the downstream receptor patched1 (PTCH1) after stimulation of the pathway via smoothened agonist (SAG). Results indicated both truncated and KO lines had an impaired response to SHH signalling, with a lack of PTCH1 upregulation following SAG treatment. With craniofacial differences being a secondary characteristic of BBS, a cranial neural crest cell (CNCC) differentiation was performed on all mutant cell lines to assess if any impairment on differentiation occurred. Neither of the BBS9 KO cell lines differentiated to form any CNCC. All truncated cell lines produced CNCC with varying efficiency and heterogeneity, which appeared to correlate with their known ciliary expression levels in their iPSC state. In summary, a platform has been created for further interrogation of the pig-productivity phenotype in a spectrum of BBS9 mutant iPSC lines to aid the goal of editing the mutation to circumvent lethality and thus improve growth and productivity in livestock.