Application of a Drosophila melanogaster model to study Familial Isolated Pituitary Adenomas syndrome pathogenesis in vivo

A phenotypically distinct subgroup of familial isolated pituitary adenoma (FIPA) families has mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene leading to young-onset acromegaly in most patients. These patients typically develop invasive pituitary adenomas, but the mechanisms by which AIP inactivation promotes pituitary tumorigenesis and an aggressive behaviour remain unknown. To date, more than 70 different AIP variants have been reported and determining the pathogenicity of missense variants is a challenging problem. The Drosophila AIP orthologue (CG1847) is located on the X chromosome and encodes a protein of similar size and structure to human protein (hAIP). I have generated CG1847 deficient flies via two methods: in vivo RNAi knockdown and imprecise excision of a transposable P-element, which generated a putative null allele of CG1847. Our data show that knockdown and knockout of CG1847 results in lethality confirming that AIP is an essential gene. To reveal the potential underlying molecular mechanisms of loss of AIP, a whole transcriptome analysis was performed in mutant versus control male larvae. This allowed us to determine gene expression profiles and to identify key pathways that are significantly altered in the mutant, and that are related to embryonic development or survival. To functionally test the homology between hAIP and CG1847, I used the Gal4/UAS system to perform rescue experiments. I subsequently tested whether wild-type hAIP, a truncated hAIP and four missense mutations identified in FIPA families could rescue the lethality of CG1847exon1_3 mutants by expressing hAIP during fly development. In this thesis were identified novel AIP features. CG1847 is a Drosophila melanogaster AIP orthologue and is essential for normal development. RNA sequencing revealed possible new underlying CG1847 molecular mechanisms as the tumour suppressor function of AIP might involve the regulation of cytoskeletal organisation. Drosophila is a useful in vivo system to study human AIP missense variants to establish pathogenicity.William Harvey Research Foundation European Society of Endocrinolog

In vivo bioassay to test the pathogenicity of missense human AIP variants

Background Heterozygous germline loss-of-function mutations in the aryl hydrocarbon receptor-interacting protein gene (AIP) predispose to childhood-onset pituitary tumours. The pathogenicity of missense variants may pose difficulties for genetic counselling and family follow-up. Objective To develop an in vivo system to test the pathogenicity of human AIP mutations using the fruit fly Drosophila melanogaster. Methods We generated a null mutant of the Drosophila AIP orthologue, CG1847, a gene located on the Xchromosome, which displayed lethality at larval stage in hemizygous knockout male mutants (CG1847exon1_3 ). We tested human missense variants of ‘unknown significance’, with ‘pathogenic’ variants as positive control. Results We found that human AIP can functionally substitute for CG1847, as heterologous overexpression of human AIP rescued male CG1847exon1_3 lethality, while a truncated version of AIP did not restore viability. Flies harbouring patient-specific missense AIP variants (p.C238Y, p.I13N, p.W73R and p.G272D) failed to rescue CG1847exon1_3 mutants, while seven variants (p.R16H, p.Q164R, p.E293V, p.A299V, p.R304Q, p.R314W and p.R325Q) showed rescue, supporting a non-pathogenic role for these latter variants corresponding to prevalence and clinical data. Conclusion Our in vivo model represents a valuable tool to characterise putative disease-causing human AIP variants and assist the genetic counselling and management of families carrying AIP variants