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.
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