Epistasis between RET and BBS mutations modulates enteric innervation and causes syndromic Hirschsprung disease

Abstract

Hirschsprung disease (HSCR) is a common, multigenic neurocristopathy characterized by incomplete innervation along a variable length of the gut. The pivotal gene in isolated HSCR cases, either sporadic or familial, is RET. HSCR also presents in various syndromes, including Shah-Waardenburg syndrome (WS), Down (DS), and Bardet-Biedl (BBS). Here, we report 3 families with BBS and HSCR with concomitant mutations in BBS genes and regulatory RET elements, whose functionality is tested in physiologically relevant assays. Our data suggest that BBS mutations can potentiate HSCR predisposing RET alleles, which by themselves are insufficient to cause disease. We also demonstrate that these genes interact genetically in vivo to modulate gut innervation, and that this interaction likely occurs through complementary, yet independent, pathways that converge on the same biological process.

Fig. 1.

RET and BBS mutations found in BBS+HSCR cases. Segregation of BBS and RET mutations in 2 BBS families with BBS±HSCR and 1 BBS+HSCR sporadic case (SB25).

Fig. 2.

In vivo assessment of the pathogenic potential of BBS4 S457I variant. (A) Zebrafish embryos coinjected with a MO against bbs4 and mRNA expressing the S457I variant exhibited defects, including short body axes, broad somites, wider and kinked notochords, and detachment of cells along the dorsal axis. (B) Compared to rescue with WT BBS4 mRNA, rescue with the mutated mRNA produced an increase in the proportion of both moderately (Class I) and severely (Class II) affected embryos, but injection of S457I mRNA alone does not produce severe defects, suggesting that the variant is functionally null. (C) HeLa cells transfected with either WT or S457I mutant BBS4 protein (red). WT protein localizes to the basal body (γ-tubulin, green), but mutant protein completely mislocalizes to form cytoplasmic aggregates.

Fig. 3.

Deletion in the RET regulatory sequence perturbs reporter gene expression in vitro. (A) Schematic representation of vectors used to assay the effect of the 11-bp deletion on reporter gene expression in vitro. WT or mutated (Δ11bp) RET enhancer sequence (green) was placed upstream of an SV40 promoter (gray) driving firefly luciferase expression (yellow). The pDSma-RET MCS + 9.7, described previously (5), was used as a positive control. (B) Relative luciferase activity of pDSma-RET vectors (depicted in A) in SK-N-SH neuroblastoma cells. Cells were lysed 48 h posttransfection, and relative luciferase activity was normalized to values obtained from the promoter alone (pDSma-Promoter). The WT RET enhancer sequence significantly decreases reporter gene expression (P = 0.001) compared with promoter alone, suggesting that it contains a putative transcription repressor-binding site. However, the vector harboring the 11-bp deletion significantly derepresses reporter gene expression compared with WT (P = 0.05) to a level statistically indistinguishable (NS) from the promoter alone, suggesting specific disruption of the repressor element binding motif. Assays were performed in triplicate wells and repeated thrice.

Fig. 4.

Genetic interaction of Ret and Bbs in zebrafish. (A) Migration of enteric neurons in 4-day-old embryos normally proceeds along the developing hindgut to reach the anus (asterisks). Embryos injected with MO against either (B) ret or (C) bbs4, (E) bbs5, or (G) bbs7 exhibit premature termination of enteric neuron migration (arrowhead). This defect is exacerbated in embryos injected with a combination of ret MO and either (D) bbs4, (F) bbs5, or (H) bbs7 MO. (I) The extent of the defect was quantified by assessment of the presence of phox2b expression in the proximal hindgut (somites 4–9) or the distal hindgut (somites 10 and beyond) as previously described (31).