Truncating loss-of-function mutations of DISP1 contribute to holoprosencephaly-like microform features in humans

Abstract

Defective function of the Sonic Hedgehog (SHH) signaling pathway is the most frequent alteration underlying holoprosencephaly (HPE) or its various clinical microforms. We performed an extensive mutational analysis of the entire human DISP1 gene, required for secretion of all hedgehog ligand(s) and which maps to the HPE 10 locus of human chromosome 1q41, as a HPE candidate gene. Here, we describe two independent families with truncating mutations in human DISP1 that resemble the cardinal craniofacial and neuro-developmental features of a recently described microdeletion syndrome that includes this gene; therefore, we suggest that DISP1 function contributes substantially to both of these signs in humans. While these clinical features are consistent with common HPE microforms, especially those linked to defective signaling by Sonic Hedgehog, we have insufficient evidence so far that functionally abnormal DISP1 alleles will commonly contribute to the more severe features of typical HPE.

Fig. 1

Protein alignment of human DISP1 and murine Disp1 proteins based on the Clustal W algorithm (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Both of these vertebrate Disp are orthologs of Drosophila disp (not shown) are predicted to share a common topology with a core 12 membrane-spanning structure (each transmembrane element is boxed to indicate the membrane embedded amino acids) including the segment between boxed transmembrane regions (TM) 2–5 comprising the sterol-sensing domain. Key differences between genetic variants of the human and murine proteins are highlighted in orange (mis-sense or in-frame deletion) or red (truncating mutations)

Fig. 1

Protein alignment of human DISP1 and murine Disp1 proteins based on the Clustal W algorithm (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Both of these vertebrate Disp are orthologs of Drosophila disp (not shown) are predicted to share a common topology with a core 12 membrane-spanning structure (each transmembrane element is boxed to indicate the membrane embedded amino acids) including the segment between boxed transmembrane regions (TM) 2–5 comprising the sterol-sensing domain. Key differences between genetic variants of the human and murine proteins are highlighted in orange (mis-sense or in-frame deletion) or red (truncating mutations)

Fig. 2

Determination of relative functional activity of genetic variants by assessment of the transporter function of Disp on a renilla-tagged Shh reporter in Drosophila S2R+ cells. a The standard dose–response curve with transfected murine DispA documents that increasing amounts of murine DispA (determined by measurement of the firefly luciferase activity on the X-axis) results in a maximum stimulation of ShhE-Rluc export of fourfold. In contrast, the murine DispB or the human DISPA fail to enhance ShhE-Rluc reporter export under these conditions (in spite of adequate expression of the N-firefly luciferase-tagged proteins). Five of the mis-sense changes incorporated within the murine DispA backbone cDNA could be studied; however, none of these variants appreciably affected the transporter-like function of the Disp test protein. b Normally, a bioactive murine Disp1 construct will enhance the export of the tagged Shh ligand by 4- to 10-fold (Ma et al. 2002). Both truncation mutations (murine construct versions mimicking W475X and Y734X) abrogate the ability of the Disp1 test molecule to influence ShhE-Rluc export. Values reported are the maximal release values obtained at a fixed input of Disp1 construct used in all three examples