Novel insights into the phenotypic spectrum and pathogenesis of Hardikar syndrome

Abstract

Purpose: Hardikar syndrome (HS, MIM #301068) is a female-specific multiple congenital anomaly syndrome characterized by retinopathy, orofacial clefting, aortic coarctation, biliary dysgenesis, genitourinary malformations, and intestinal malrotation. We previously showed that heterozygous nonsense and frameshift variants in MED12 cause HS. The phenotypic spectrum of disease and the mechanism by which MED12 variants cause disease is unknown. We aim to expand the phenotypic and molecular landscape of HS and elucidate the mechanism by which MED12 variants cause disease.

Methods: We clinically assembled and molecularly characterized a cohort of 11 previously unreported individuals with HS. Additionally, we studied the effect of MED12 deficiency on ciliary biology, hedgehog, and yes-associated protein (YAP) signaling; pathways implicated in diseases with phenotypic overlap with HS.

Results: We report novel phenotypes associated with HS, including cardiomyopathy, arrhythmia, and vascular anomalies, and expand the molecular landscape of HS to include splice site variants. We additionally demonstrate that MED12 deficiency causes decreased cell ciliation, and impairs hedgehog and YAP signaling.

Conclusion: Our data support updating HS standard-of-care to include regular cardiac imaging, arrhythmia screening, and vascular imaging. We further propose that dysregulation of ciliogenesis and YAP and hedgehog signaling contributes to the pathogenesis of HS.

Keywords: Cilia; Hardikar syndrome; Hedgehog; MED12; YAP.

Figure 1:

Schematic of the MED12 protein (NP_005111.2) depicting variants identified in individuals with HS. Red and underlined denotes recurrent variants.

Figure 2:

(A) RPE cells were grown in complete medium for 24 hours and then switched to fresh complete medium (control) versus serum-free medium to promote ciliogenesis. RNA was harvested after 24 hours for qPCR. (B) RPE cells were serum starved for 48 hours. Medium was replaced with fresh, serum-free medium (continued ciliogenesis) or complete medium (cell cycle entry, ciliary disassembly) and RNA was harvested for qPCR after 24 hours. (C) RPE cells were transfected with control or MED12 siRNA, serum starved for 48 hours to promote ciliogenesis and RNA was harvested for qPCR (upper panel) and protein was harvested for immunoblot (lower panel). (D - G) RPE cells were plated in an 8-well chamber slide, transfected with control versus MED12 siRNA, and serum starved for 48 hours to promote ciliogenesis. Cilia were visualized using antibodies against acetylated alpha tubulin (green) and ARL13B (red). (H) RPE cells were plated and grown in complete medium for 24 hours and then transfected with control versus MED12 siRNA. After 24 hours, cells were replated at equal density, serum starved for 24 hours, and cells were counted via trypan blue staining. (I) RPE cells were transfected with control or MED12 siRNA as indicated. Cells were serum starved for 24 hours to promote ciliogenesis, then treated with DMSO (control) or 100 nM SAG for 24 hours, and RNA was harvested for qPCR (n = 3 experiments, analyzed via student T-test (panels A – H) or two-way ANOVA (panel I) in GraphPad Prism; **** P < 0.0001; ** P < 0.01).

Figure 3:

(A,B) RPE cells were plated and grown in complete medium for 24 hours then transfected with control versus MED12 siRNA. RNA was harvested after 72 hours for qPCR. (C) RPE cells were plated and grown in complete medium for 24 hours and then transfected with control versus MED12 siRNA. After 24 hours cells were co-transfected with plasmids encoding renilla driven by a thymidine kinase promoter and firefly luciferase driven by a TEAD-responsive promoter. Fluorescence activity was measured using the dual luciferase reporter assay after 48 hours. (D) RPE cells were plated and grown in complete medium for 24 hours then transfected with control versus MED12 siRNA. RNA was harvested after 72 hours for qPCR. (E – H) RPE cells were plated and grown in complete medium for 24 hours then transfected with control versus MED12 siRNA. Protein was harvested after 72 hours in 1x LDS for immunoblot. (n = 3 experiments, analyzed via student T-test in GraphPad Prism; **** P < 0.0001; *** P < 0.001; ** P < 0.01).

Figure 4:

Proposed model for the pathogenesis of HS (A) Hypophosphorylated YAP translocates into the nucleus and binds the transcription factor TEAD to activate YAP downstream target genes that facilitate organogenesis and tubulogenesis. MED12 promotes YAP signaling. MED12 additionally promotes transcription of genes involved in ciliogenesis. (B) In the absence of MED12, YAP pathway activation is impaired, leading to dysregulated organogenesis and tubulogenesis (aortic coarctation, vascular defects, clefting, biliary defects, ureteral defects). Furthermore, ciliogenesis genes are not induced, leading to dysregulated hedgehog and ciliary signaling.