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

Abstract

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 (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.

Keywords: AIP; FIPA; drosophila melanogaster; pathogenic genetic variant; pituitary adenoma.

Figure 1

Comparison of Drosophila CG1847 and human AIP proteins. (A) The human and Drosophila proteins are similar; they share 120 identical amino acids, 80 strongly conserved and 34 weakly conserved amino acids. Stars indicate identity, and colons and dots indicate high and low similarity amino-acids, respectively. (B) A three-dimensional predicted model of CG1847 indicates that the Drosophila protein has a similar structure to its human orthologue, with an N-terminal PPIase domain, three pairs of conserved antiparallel alpha-helices forming the tetratricopeptide repeat domains (TPRs) and the final extended α-helix, α−7. AIP, aryl hydrocarbon receptor-interacting protein gene.

Figure 2

The lethality of CG1847 mutants can be rescued by expression of wild-type (wt) CG1847 under the control of its own promoter. (A) Schematic diagram of the wt CG1847 (i), CG1847^{exon1 _3} (ii), CG1847 rescue construct and (iii) transgenic generated human AIP rescue construct (iv). The diagram represent the mRNA, with red boxes representing coding region and white boxes representing 5′ and 3′ untranslated regions. (ii) The CG1847 mutant was generated by imprecise P-element excision of P-CG1847^G1839 (EP in the figure). (iii) Schematic representation of transgenic animals with genomic rescue construct containing the regulatory and coding regions of CG1847 inserted on the second chromosome. (iv) Schematic representation of transgenic animals with the AIP cDNA rescue construct inserted on the second chromosome. (B) Transgenic males carrying the CG1847 genomic rescue construct and balanced over CyO were crossed to the CG1847^exon1_3 heterozygous females and examined for their ability to rescue male lethality. Segregation of alleles and the possible combinations are shown in the lateral panels. Panel (i): male carrying the mutant CG1847 allele inherited from their mothers rescued by the wt CG1847 allele on the second chromosome. Panel (iii): males are not viable as they carry the CG1847 mutant allele and lack the genomic rescue construct from the paternal chromosome 2. Panels (ii) and (iv) depict male progeny lacking the CG1847 mutation. (C) Statistical analysis of rescue experiments with the CG1847 genomic rescue construct (n=4). The associated letters (panels i–iv) correspond to the phenotypes depicted in Figure 2B. X-axis labels CG1847-1M and CG1847-3M represent two different transgenic stocks carrying the rescue construct. Error bars represent SE of the mean. Asterisks indicate statistical significance as determined by Student’s t-test (**P<0.01). AIP, aryl hydrocarbon receptor-interacting protein gene; bp, base pairs.

Figure 3

Human AIP functionally complements the Drosophila orthologue. (A) Transgenic males with hAIP rescue construct were crossed to heterozygous CG1847 deficient females. The ubiquitous actin-Gal4 driver was used to drive the expression of the UAS-hAIP constructs during fly development. Panel (i) Images of F1 viable rescued males. Males expressing wt hAIP in the CG1847 mutant background (mutant CG1847 allele inherited from their maternal X chromosome and expression of the hAIP transgene on chromosome 2); panels (iii) and (v): males inheriting the mutant CG1847 allele and lacking hAIP expression are not viable. These two genotypes also serve as internal negative controls. Panels (ii) and (iv): males lacking the CG1847 mutation are viable. (B) Schematic diagram of UAS-hAIP constructs. Top, wt UAS-hAIP. Second line: artificially generated truncated hAIP lacking the seventh alpha helix. Transgenic lines for 11 different hAIP missense mutations (p.R16H, p.C238Y, p.A299V, p.R304Q, p.I13N, p.W73R, p.Q164R, p.G272D, p.E293V, p.R314W and p.R325Q) were also generated. The approximate position of amino acid changes introduced are indicated with an asterisk. Protein domains are indicated by the colour code shown below the deletion construct assembly (the colours of the domains match what is shown in the 3D model, Figure 1). (C) Quantitative analysis of in vivo rescue experiments using hAIP missense variants (n=6). Successful rescue of lethality was scored as the presence of males with the genotype CG1847^exon1_3/Y; actin-Gal4/UAS-hAIP, which lacks endogenous CG1847. Error bars represent SE of the mean. Significant differences are indicated by asterisks (****P<0.0001). AIP, aryl hydrocarbon receptor-interacting protein gene; TPR, tetratricopeptide repeat; wt, wild type.

Figure 4

The different AIP constructs, wild-type human AIP (hAIP-wt) and various missense variants, show equivalent expression levels when ubiquitously expressed using the actin-Gal4 driver. CG1487 protein expression was not detected in wild-type flies (w^iso), suggesting the anti-AIP antibody is specific for the human protein and does not recognise endogenous CG1487. β-tubulin was used as loading. Surviving males I13N*, W73R* and G272D* are viable as they have endogenous CG1487 but no hAIP due to non-disjunction (further details of the non-disjunction phenomenon is presented in online supplementary material 1). AIP, aryl hydrocarbon receptor-interacting protein gene.