The LRBA variant identified does not affect splicing nor its enhancing ability, and is likely not involved in the regulation of MAB21L2 expression. (A) Exon trap assay showed that splicing of exon 20 of LRBA is not affected by the presence of the variant identified (c.2444A>G), as similar-size bands were obtained for WT and Mut constructs. E, empty vector; M, 1Kb+ DNA marker (Invitrogen); Un, untransfected cells. (B) Schematic overview of the genomic region of LRBA with MAB21L2 as a nested pair (located in intron 42 of LRBA), and their respective positions in the human genome (hg19). The variant in exon 20 of LRBA is located 288.6 Kb away from the start site of MAB21L2. (C) Immunohistochemistry performed with an HuC/Elavl3 antibody in control and mab21l2 mutant zebrafish embryos, showed that the absence of mab21l2 leads to an overall reduction in the numbers of enteric neurons, and aganglionosis is detected in the gut. (D) Luciferase assays performed to evaluate a possible enhancer effect of the LRBA variant (c.2444A>G) showed that although exon 20 has enhancer activity when coupled to an SV40 promoter (SV40-P), no difference in luciferase activity could be detected between LRBA WT and LRBA Mut (c.2444A>G) constructs. SV40-E construct was used as a negative control, and a RET intronic enhancer element (RET-WT) was used as a positive control.

Variants in RET and IHH have a pathogenic nature. (A) Western blot analysis of HEK293 cells transiently expressing pCMV-RET-WT and pCMV-RET-Mut showed that the RET variant identified (c. 1196C>T, p.RET-P399L), leads to a reduction of glycosylated protein, as well as a reduction in the levels of phosphorylated RET. β-actin was used as loading control and GFP as transfecting control. (+) presence and (−) absence of GDNF (50 ng/mL). UT, untransfected. (B) Western blot analysis of HEK293 cells transiently expressing IHH-WT-FLAG and IHH-Q51K-FLAG showed no difference in the expression of IHH precursor (∼46 kDa). (C) qreal time-PCR performed in fibroblasts grown in the presence of conditioned medium containing IHH-WT or IHH-Q51K secreted proteins, showed that cells stimulated with the mutant IHH have reduced expression of GLI1 when compared with cells stimulated with the WT protein. Purmorphamine (PUR+), an activator of the Hh signaling, was used as a positive control. (D) Analysis of the uninjected control and ihh morphant embryos at 120 hpf showed that the absence of ihh led to curvature of the body, smaller eyes, craniofacial abnormalities, and a loss of swim bladder. Moreover, a decreased number of enteric neurons was observed in morphant embryos after staining with an Elavl3-specific antibody. * marks the anus of the fish.

mab21l2 expression pattern in zebrafish. In situ hybridization showing that mab21l2 has a strong expression in the hindbrain (black arrows) and pharyngeal arches (white arrows) through all time points (24–96 hpf). From 48 hpf onward, a strong expression is also detected in the gut mesoderm (*).

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