Reassessing the role of the p.(Arg304Gln) missense AIP variant in pituitary tumorigenesis

Abstract

Objective: Heterozygous germline loss-of-function variants in AIP are associated with young-onset growth hormone and/or prolactin-secreting pituitary tumours. However, the pathogenic role of the c.911G > A; p.(Arg304Gln) (R304Q) AIP variant has been controversial. Recent data from public exome/genome databases show this variant is not infrequent. The objective of this work was to reassess the pathogenicity of R304Q based on clinical, genomic, and functional assay data.

Design: Data were collected on published R304Q pituitary neuroendocrine tumour cases and from International Familial Isolated Pituitary Adenoma Consortium R304Q cases (n = 38, R304Q cohort). Clinical features, population cohort frequency, computational analyses, prediction models, presence of loss-of-heterozygosity, and in vitro/in vivo functional studies were assessed and compared with data from pathogenic/likely pathogenic AIP variant patients (AIPmut cohort, n = 184).

Results: Of 38 R304Q patients, 61% (23/38) had growth hormone excess, in contrast to 80% of AIPmut cohort (147/184, P < .001). R304Q cohort was older at disease onset and diagnosis than the AIPmut cohort (median [quartiles] onset: 25 y [16-35] vs 16 y [14-23], P < .001; median [quartiles] diagnosis: 36 y [24-44] vs 21 y [15-29], P < .001). R304Q is present in gnomADv2.1 (0.31%) and UK Biobank (0.16%), including three persons with homozygous R304Q. No loss-of-heterozygosity was detected in four R304Q pituitary neuroendocrine tumour samples. In silico predictions and experimental data were conflicting.

Conclusions: Evidence suggests that R304Q is not pathogenic for pituitary neuroendocrine tumour. We recommend changing this variant classification to likely benign and do not recommend pre-symptomatic genetic testing of family members or follow-up of already identified unaffected individuals with the R304Q variant.

Keywords: AIP; FIPA; acromegaly; genetic variant; gigantism; prolactinoma.

Graphical Abstract

Figure 1.

Clinical presentation and genetic findings in R304Q patients from the IFC cohort. A) Family trees of the 6 FIPA families with the AIP R304Q variant and of 3 representative simplex patients within the IFC. B) Affected members of Family 1. The proband (Patient 2) was diagnosed with gigantism at age 17 years, caused by a pituitary macroadenoma with cavernous sinus extension. Her mother (Patient 1) was diagnosed with acromegaly at the age of 44 years; her magnetic resonance imaging showed an invasive pituitary macroadenoma (maximum diameter: 46 mm) with cavernous sinus and suprasellar extension, resulting in a visual field defect. Written informed consent for publication of their clinical details and clinical images was obtained from the patients. C) Sanger sequencing of the R304Q surrounding sequence displayed no LOH in DNA extracted from the tumour, compared with germline DNA. D) Electron microscopy images of one somatotrophinoma associated with R304Q (Case 16). Tumour cells have secretory granules that vary from sparse to numerous and vary in size, shape, and electron density (d1). The cells display a prominent Golgi apparatus (d2), with prominent profiles of rough endoplasmic reticulum (d3). The secretory granules vary in size, shape, and electron density. Occasional cells have cytoplasmic fibrous bodies, as seen centrally (d4).

Figure 2.

AIP R304Q protein structure and functional assays. A) Three dimensional and B) linear structure of AIP protein with close-up of location of R304Q (insert). The AIP co-chaperone protein (330 amino acids) has a peptidyl-prolyl ci-trans isomerase (PPIase)-like domain at the N-terminus, 3 tetratricopeptide domains arranged as antiparallel alpha helices, and a final α−7 helix at the C-terminus. The helical structures enable AIP to interact with numerous partners, including HSP90, AHR, and phosphodiesterases. C) Position 304 is conserved and occupied by the positively charged residues arginine (R) or lysine (K) in the multiple sequence alignment. D) Model of interaction of AIP variant R304Q with Tomm20 protein. AIP is presented in grey, and part of the Tomm20 AQSLAEDDVE-peptide in yellow. Residue Arg304 is presented in green. E) R304Q maintains its interaction with HSPA8. HEK293 cells were co-transfected with pcDNA3.0-Myc-AIP_ R304Q and pSF-CMV-NH2-HA-EKT-NcoI-HSPA8, and immunoprecipitation was performed using anti-Myc or anti-HA mouse antibodies, or mouse IgG. Eluates were resolved by denaturing polyacrylamide gel electrophoresis, followed by Western blot, using anti-Myc and anti-HA antibodies. Top arrow: HA-HSPA8, bottom arrow: Myc-AIP. Arrowhead: heavy chain of mouse immunoglobulins. F) Fluorescence double immunostaining of GH and PDE4A8 in normal pituitary, a tumour without AIP variant (no AIP Var), a tumour with R304* variant and a tumour with R304Q variant; scale bar 25 µm. ****P < .0001 Kruskal–Wallis followed by Dunn's test. G) Fluorescence double immunostaining of GH and PDE4A4 in normal pituitary, a tumour without AIP variant (No AIP Var), a tumour with R304* variant and a tumour with R304Q variant; scale bar 25 µm. ****P < .0001 Kruskal–Wallis followed by Dunn's test. H) Half-life of wild-type AIP and of the AIP variant proteins R304Q and R304*, overexpressed in HEK293 cells. The degradation speed of the R304Q variant (K = .0118) was not significantly different to that of the wild-type protein (K = 0.0145, P = .5644) while the variant R304* showed rapid degradation compared with the wild-type protein (K = 0.1183, P < .0001). The representative WB images show bands for Myc-AIP wild-type and R304Q (39 kDa), Myc-AIP R304* (35.8 kDa) and ACTB loading control (41.7 kDa); data extracted from. I) Cyp1a1 relative mRNA levels in Aip shRNA-KD GH3 cells treated with 10 nm FICZ for 5 hours (EV: empty vector, n = 3; WT-AIP: wild-type AIP, n = 6; R304*, n = 9; R304Q, n = 6). Error bars indicate SEM. ANOVA followed by Tukey–Kramer honest significant difference post-hoc test (**P < .01). MWM, molecular weight marker; IP, immunoprecipitation.