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 viv o 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 ( CG1847 exon1_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 CG1847 exon1_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 CG1847 exon1_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.