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Case Reports
. 2012 Dec 7;91(6):1122-7.
doi: 10.1016/j.ajhg.2012.10.013. Epub 2012 Nov 15.

Recurrent de novo mutations in PACS1 cause defective cranial-neural-crest migration and define a recognizable intellectual-disability syndrome

Janneke H M Schuurs-Hoeijmakers  1 , Edwin C Oh, Lisenka E L M Vissers, Mariëlle E M Swinkels, Christian Gilissen, Michèl A Willemsen, Maureen Holvoet, Marloes Steehouwer, Joris A Veltman, Bert B A de Vries, Hans van Bokhoven, Arjan P M de Brouwer, Nicholas Katsanis, Koenraad Devriendt, Han G Brunner
Affiliations
Case Reports

Recurrent de novo mutations in PACS1 cause defective cranial-neural-crest migration and define a recognizable intellectual-disability syndrome

Janneke H M Schuurs-Hoeijmakers et al. Am J Hum Genet. .

Abstract

We studied two unrelated boys with intellectual disability (ID) and a striking facial resemblance suggestive of a hitherto unappreciated syndrome. Exome sequencing in both families identified identical de novo mutations in PACS1, suggestive of causality. To support these genetic findings and to understand the pathomechanism of the mutation, we studied the protein in vitro and in vivo. Altered PACS1 forms cytoplasmic aggregates in vitro with concomitant increased protein stability and shows impaired binding to an isoform-specific variant of TRPV4, but not the full-length protein. Furthermore, consistent with the human pathology, expression of mutant PACS1 mRNA in zebrafish embryos induces craniofacial defects most likely in a dominant-negative fashion. This phenotype is driven by aberrant specification and migration of SOX10-positive cranial, but not enteric, neural-crest cells. Our findings suggest that PACS1 is necessary for the formation of craniofacial structures and that perturbation of its functions results in a specific syndromic ID phenotype.

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Figures

Figure 1
Figure 1
Photographs and Genetic Data of Two Unrelated Individuals with an Identical De Novo Mutation in PACS1 (A) Upper photograph: individual 1 at 4 years of age with a low anterior hairline, highly arched eyebrows, synophrys, hypertelorism with downslanted palpebral fissures, long eyelashes, a bulbous nasal tip, a flat philtrum with a thin upper lip, downturned corners of the mouth, diastema of the teeth, and low-set ears. Bottom photograph: individual 2 at 12 years of age. Note the remarkable facial similarity. (B) Sequence reads from exome sequencing and chromatograms of Sanger confirmation show the identical de novo occurrence of the c.607C>T mutation in PACS1 (RefSeq NM_018026.2) in individuals 1 and 2. (C) Protein structure of PACS1. The position of the p.Arg203Trp substitution is indicated in the furin (cargo)-binding region (FBR) of the protein and is directly adjacent to the CK2-binding motif.
Figure 2
Figure 2
In Vivo Functional Characterization of the p.Arg203Trp Substitution in PACS1 (A) Alcian-blue staining of 4-day-old zebrafish larvae expressing either 50 pg wild-type (c.607C [p.Arg203]) or 50 pg mutant (c.607T [p.Trp203]) PACS1 RNA. Left panel: craniofacial cartilaginous structures visualized in both lateral and ventral views of the embryo. Right panel: craniofacial phenotypes in embryos expressing wild-type PACS1, mutant PACS1, and both wild-type and mutant PACS1 are quantified. White arrows and asterisks highlight Meckel’s cartilage in the lateral and ventral perspectives of the embryos. Human wild-type and mutant PACS1 mRNA was transcribed in vitro with a mMESSAGE mMACHINE SP6 Kit (Ambion), and 0.5 nl was microinjected into 2- to 4-cell-stage zebrafish embryos. (B) Imaging of 4-day-old sox10::eGFP zebrafish larvae expressing either 50 pg wild-type or mutant PACS1 RNA. Left panel: migration of eGFP-labeled cranial-neural-crest cells (CNCCs). Right panel: CNCC-migration phenotype scored in embryos expressing wild-type PACS1, altered PACS1, and both wild-type and altered PACS1.
Figure 3
Figure 3
In Vitro Functional Characterization of the p.Arg203Trp Substitution in PACS1 (A) Localization of GFP-tagged wild-type and altered PACS1 in transfected ARPE-19 cells grown to confluence and stained with a GFP antibody. ARPE-19 cells were grown in Dulbecco’s Modified Eagle Medium and Ham’s F-12 Nutrient 1:1 mixture (DMEM/F-12, Invitrogen) with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. Transfection of wild-type and mutant PACS1 plasmids was carried out with FuGene6 Transfection Reagent (Roche). Cells were fixed with 4% paraformaldehyde 72 hr after transfection and were probed with a GFP antibody (Santa Cruz, sc-8334) and a secondary antibody, Alexa Fluor 488 IgG (Invitrogen). (B) Quantification of wild-type and p.Trp203 PACS1 stability in transfected cells treated with cycloheximide (CHX). The mean measurement of triplicate experiments is shown, and the error bar represents the SEM. HEK 293FT cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) containing 10% FBS (Invitrogen) and 2 mM L-glutamine (Invitrogen). Cells were treated with 50 mM CHX (Sigma) for 6 hr and harvested in co-IP buffer. (C) Immunoprecipitation of GFP-tagged wild-type and altered PACS1 and V5-tagged TRPV4v1 (RefSeq NM_021625) and TRPV4v2 (RefSeq NM_147204). HEK 293 cells were transfected with tagged constructs and harvested in co-IP buffer after 48 hr. Immunoprecipitation was performed with a GFP antibody and immunoblotted with a V5 antibody.
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