Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD)

Abstract

KAL1 is implicated in 5% of Kallmann syndrome cases, a disorder which genotypically overlaps with septo-optic dysplasia (SOD). To date, a reporter-based assay to assess the functional consequences of KAL1 mutations is lacking. We aimed to develop a luciferase assay for novel application to functional assessment of rare KAL1 mutations detected in a screen of 422 patients with SOD. Quantitative analysis was performed using L6-myoblasts stably expressing FGFR1, transfected with a luciferase-reporter vector containing elements of the FGF-responsive osteocalcin promoter. The two variants assayed [p.K185N, p.P291T], were detected in three females with SOD (presenting with optic nerve hypoplasia, midline and pituitary defects). Our novel assay revealed significant decreases in transcriptional activity [p.K185N: 21% (p < 0.01); p.P291T: 40% (p < 0.001)]. Our luciferase-reporter assay, developed for assessment of KAL1 mutations, determined that two variants in females with hypopituitarism/SOD are loss-of-function; demonstrating that this assay is suitable for quantitative assessment of mutations in this gene.

Keywords: Females; KAL1; Kallmann syndrome; Luciferase assay; Septo-optic dysplasia.

Supplementary Fig. 1

Schematic representation of expression and reporter vectors used for novel application of our luciferase reporter assay for functional assessment of KAL1 variants in vitro. A, Full length human KAL1 (NM_000216) had previously been cloned into the SalI (5′) and BamHI (3′) restriction sites of the pEGFP-N1 expression vector (Clontech) (Hu et al., 2009). In the presence of the mutated KAL1 stop codon, the encoded protein anosmin-1 is conjugated to GFP at the C-terminal. Empty pEGFP-N1 vector which expresses GFP alone was used as control. B, The p6xOSE2-Luc reporter vector was produced as previously described (Kim et al., 2003, Park et al., 2010). Six identical osteoblast-specific core binding sequences (bold) (OSE2) of the FGF responsive osteocalcin promoter were synthesised with XmaI overhangs at each 5′ end of the oligonucleotides and ligated. Multimers were then cloned into the SmaI/XmaI restriction site of the pGL2-promoter reporter vector (Promega). C, Luciferase activity stimulated by the KAL1 expression vector via the p6xOSE2-Luc reporter vector was normalised to the constitutively active Renilla luciferase reporter pRL-SV40 (Promega).

Fig. 1

Electropherogram representation of two heterozygous variants found in KAL1 and their conservation across multiple species. Variant c.555G > C, p.K185N (A; mutant sequence on bottom indicated by arrow) was detected in a female patient with septo-optic dysplasia, characterised by optic nerve and pituitary defects. The variant was highly conserved from mammals to zebrafish (B). Variant c.871C > A, p.P291T (C; mutant sequence on bottom indicated by arrow) was detected in two sisters with septo-optic dysplasia characterised by optic nerve and pituitary defects, as well as cavum septum pellucidum. The variant was highly conserved across mammals (D).

Fig. 2

Ribbon representation of the six domain solution structure model of anosmin-1, N-terminal on left. Beta-strand ribbons in yellow, helix ribbon in red and turns in blue. The domains are labelled as follows: CR, cysteine rich region; WAP, whey acidic protein-like, FnIII, fibronectin type III. The p.K185N, located between WAP and FnIII.1, and p.P291T, located between FnIII.1 and FnIII.2, variations are indicated in space filling representation for the N and T residues. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Qualitative functional analysis of anosmin-1 protein localisation/expression in transfected Cos7 cells. A-D, expression constructs encoding wt KAL1 (A) or variant p.K185N or p.P291T proteins (B and C/D (low magnification) respectively) conjugated to GFP were transfected into Cos7 cells. Two days after transfection, cells were fixed for immunocytochemical analysis or lysed for protein extraction prior to western blot analysis (E). For the immunocytochemical stains, anosmin-1 (green), was co-localised with β-catenin of the adherens junction (red) to highlight cellular membranes. Cell nuclei were highlighted with DAPI (blue). Secreted anosmin-1 is indicated by arrows and retained protein is indicated by asterisks. E, western blot analysis was conducted on protein extracted from cell lysates or culture media. Bands corresponding to anosmin-1 are indicated by arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Quantitative functional analysis of anosmin-1 variants. A, Schematic representation of the FGFR pathway which our luciferase reporter vector (p6xOSE2-Luc) exploits. Binding of FGF2 ligand to FGFR1 stimulates dimerisation of the receptor and subsequent signalling cascades. These signals are enhanced through anosmin-1 binding to the receptor, which can be positively mediated by heparan sulfate. B, In order to validate our functional assay, FGFR1-expressing L6-myoblasts were transfected with our luciferase reporter and then treated with increasing doses of rhFGF2. C and D, FGFR1-expressing L6-myoblasts were then transfected with empty vector (labelled as GFP) or wt or variant KAL1-GFP conjugated constructs as well as our luciferase reporter and Renilla luciferase internal control. Cells were then treated with rhFGF2 with (D) or without (C) heparin (a type of heparan sulfate). The left column of each graph labelled ‘cells,’ were untransfected cells, still treated with 1 nM rhFGF2 ± 1 μg/ml heparin. All treatments were done in triplicate, with cultures repeated three times in total. Data are presented as mean ± SD. ns = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Note that panel A is adapted from Hu and Bouloux (2011).

Fig. 5

In situ hybridisation analysis of KAL1 expression in the developing human embryo at 6 weeks (Carnegie stage 16) and 8 weeks (Carnegie stage 23) gestation. A, KAL1 mRNA transcripts were observed transiently in the dorsal aspect of Rathke's pouch (primordial anterior pituitary gland) at 6 weeks (arrowheads). B, Expression was also observed in the developing neural tube (arrows heads in B and arrow in C). C, Strong localised expression was consistently observed in the dorsal aspect of the developing otic vesicles (OV, arrowhead). D, At week 8, KAL1 mRNA transcripts were observed in the sulcus of the ventricular zone of the 3rd ventricle where the neural progenitors reside (labelled as 3V, arrowheads in B′), in the vicinity of the hypothalamus. No expression was detected in the pituitary (asterisk). B′ and C′ are enlarged images from D. C′ shows KAL1 mRNA expression in the basal ganglion (arrowheads). E, KAL1 was also observed in the developing inner ear (IE) that will form the cochlea (arrowheads). Abbreviations: 3V, third ventricle; DNT, dorsal neural tube; DRG, dorsal root ganglia; IE, inner ear; NT, neural tube; OC, oral cavity; OV, otic vesicle; RP, Rathke's pouch. Scale bars from A–D are 200 μm.