Twigg SR , Forecki J , Goos JA , Richardson IC , Hoogeboom AJ , van den Ouweland AM , Swagemakers SM , Lequin MH , Van Antwerp D , McGowan SJ , Westbury I , Miller KA , Wall SA , WGS500 Consortium , van der Spek PJ , Mathijssen IM , Pauws E , Merzdorf CS , Wilkie AO .

MC_UU_12010/6 Medical Research Council , 093329 Wellcome Trust , 102731 Wellcome Trust , Department of Health, MRC_MC_UU_12010/6 Medical Research Council , 102731/Z/13/Z Wellcome Trust , Wellcome Trust , 16459 Cancer Research UK

           CRANIOSYNOSTOSIS 4; CRS4
           CRANIOSYNOSTOSIS 2; CRS2
           CRANIOSYNOSTOSIS 3; CRS3

	Figure 1. Clinical and Radiological Phenotype of Individuals with ZIC1 Mutations(A) Subject 1, age 7 weeks (left) and 8 years (right).(B) Subject 2, age 23 years.(C) Subject 3, age 5 months.(D) CT head scan of subject 3 (age 5 months) showing bicoronal synostosis, a large wormian bone (arrow) in the position of the anterior fontanelle, and an ossification defect in the sagittal suture (arrowhead).(E) Subject 4, age 7 months.(F) CT head scan of subject 4 (age 2.5 months). Note asymmetric skull shape associated with bilateral coronal and right lambdoid suture fusion (arrow). Sections of the metopic and sagittal sutures remain widely patent (arrowheads).(G) MRI brain scan (T2 image) of subject 4, age 16 months. Note short, broad corpus callosum, peaked tentorium cerebelli (arrow), and hypoplasia of the pons (arrowhead), cerebellar vermis, and cerebellar hemispheres.(H) Pedigree of family 5.(I) Subject 5:III.6, age 2 months.(J) MRI brain scan (T2 image) of subject 5:III.6, age 13 years. Abnormal features show a similar pattern to those present in subject 4.
	Figure 2. Molecular Genetic Analysis of Individuals with ZIC1 Mutations(A) Cartoon showing exon organization (white boxes denote non-coding regions) and previously identified conserved domains (Zic opa conserved motif [ZOC], zinc finger N-flanking conserved region [ZF-NC], and five zinc fingers [1–5, blue boxes]) of human ZIC1.16 Also indicated is the C-terminal NEWYV motif conserved in all family members except ZIC4. Above the cartoon are the positions of the five independent ZIC1 mutations described in this report, and dideoxy-sequence traces showing comparison of normal sequence (above) and mutant sequence (below). Note, in the case of subject 3, the mutation was not evident in the DNA sample (sourced from scalp fibroblasts; not shown) originally analyzed; however, in the exome sequence of DNA sourced from blood of the same individual, 63 of 183 (34%) reads showed the c.1204G>T mutation, which is also readily apparent on the dideoxy sequence. The relative heights of mutant and wild-type peaks differ between samples from subject 3 and subject 2, who is constitutionally heterozygous for the identical mutation, corroborating that in subject 3 the mutation is present in high-level mosaic state.(B) Agarose gel analysis of ZIC1 cDNA obtained from RNA extracted from scalp fibroblasts of subject 1 and digested with BfaI. The fragments yielded by digestion of the mutant allele are indicated with asterisks.(C) Amino acid sequence encoded by 3′-terminal exon of ZIC1 and comparison with the paralogous human proteins ZIC2-ZIC5, showing conservation including the NEWYV motif (bracket). The end of the fifth zinc finger (Zn5) is shown above the sequence, as are the positions of the four different pathogenic variants (red symbols) (triangle, nonsense; circle, missense) identified in this study. The positions at which the Xenopus constructs zic1ΔC2 and zic1ΔC are truncated, relative to the human sequence, are indicated by blue arrows (note zic1ΔC2 is equivalent to p.Gln389∗). Additional human constructs tested in the Xenopus assay are indicated by green symbols.
	Figure 3. Analysis of Consequences of ZIC1 Mutations in Xenopus Embryos(A) Cartoon showing the structure and nomenclature of the cDNA constructs used in the experiment. The five zinc finger domains are highlighted in blue.(B) Xenopus en-2 expression after microinjection of ZIC1 construct RNA (right side of each embryo). Arrows indicate widened, increased, or shifted en-2 expression in three representative embryos.(C) Quantification of effects of ZIC1 mutations on en-2 expression. The numbers of embryos used in each experiment are indicated in parentheses. Statistical comparisons between full-length Xenopus zic1 and human ZIC1 and between ZIC1 and ZIC1-p.Glu299∗ were not significant (NS), whereas comparisons between all other mutants and corresponding full-length constructs were highly significant (p < 10−8).
	Figure 4. Expression of Zic1 in E11.5–E12.5 Mouse Embryos Analyzed by RNA In Situ Hybridization and Comparison with En1Panels show comparison of Zic1 (A, B) and En1 (C, D) expression at E11.5 (A, C) and E12.5 (B, D). In each case the top panel shows the whole embryo and plane of sections 1, 2, and 3, which are illustrated in the three respective lower panels (e, eye; t, telencephalon; d, diencephalon; m, mesencephalon; f, forebrain; circles indicate relative position of the eye). Note strong expression of Zic1 in supra-orbital region (open arrow) at E11.5; other areas of expression are midbrain-hindbrain boundary (closed arrow), neural tube, and limb mesenchyme (A). At E12.5 Zic1 expression is seen mainly in the cephalic mesenchyme, just posterior to the eye (B). By comparison, at E11.5 the expression of En1 expression is relatively weak in the supraorbital region (stronger expression is seen in the apical ectodermal ridge of the limb and in the somitic mesoderm). By E12.5 En1 expression has increased in the cephalic mesoderm.

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