Detailed clinical features for all 3MC described patients.

<p>Detailed clinical features for all 3MC described patients.</p

CL-L1 regulates cell migration and act as migratory chemoattractant.

<p>A. Experimental design for cell migration assay and invasion rate. Agarose spots containing CL-L1 and/or CL-K1 were attached to the glass and HeLa cells seeded. After 24 hours pictures were taking on different spots. The area covered inside the spot by the cells (advance front) was measured and normalised for the length of the perimeter of the spot. B. Representative pictures of agarose spots containing PBS, BSA and CL-L1 with cells invading 48 hours after seeding after. Note how HeLa cells are attracted and invade the agarose sport containing CL-L1. C. Quantification of the invasion ratio. HeLa cells when exposed to CL-L1 cells were more attracted to invade the agarose spots than PBS controls. D. ELISA results in HeLa and HEK293 cell pellets and supernatants after <i>COLEC10</i> mutant construct transfections. <i>COLEC10</i> Wild-type (<i>COLEC10</i>^WT) and three <i>COLEC10</i> mutations (<i>COLEC10</i>^{<i>ArgXTer</i>}, <i>COLEC10</i>^{Gly77Glufs*66} and <i>COLEC10</i>^Cys176Trp) cDNAs where cloned and transfected in HEK293 and HeLa cells. Untransfected cells C(-) were used as a control to prove no endogenous CL-L1 was affecting the readings. Concentration of expressed CL-L1 was tested by ELISA in cells extracts and cell supernatant. CL-L1 was found in <i>COLEC10</i>^WT and <i>COLEC10</i>^Cys176Trp cell extracts in both cell types, but no CL-L1 was expressed after transfecting <i>COLEC10</i>^{<i>ArgXTer</i>} or <i>COLEC10</i>^{Gly77Glufs*66} constructs or in the untransfected C(-) cells. <i>COLEC10</i>^WT and <i>COLEC10</i>^Cys176Trp showed very similar levels of CL-L1 expression in HeLa and HEK293 cell types, with slightly higher expression in <i>COLEC10</i>^Cys176Trp, (2518 ng/ml HeLa and 1302 ng/ml HEK293) versus <i>COLEC10</i>^WT (1823 ng/ml HeLa and 632 ng/ml HEK293). However, the levels of CL-L1 were undetectable in the supernatant of <i>COLEC10</i>^Cys176Trp transfections, in contrast with <i>COLEC10</i>^WT transfected cells (200 ng/ml HeLa and 390 ng/ml HEK293)</p

New <i>COLEC10</i> mutations found in families MC19 and MC25

<p>New <i>COLEC10</i> mutations found in families MC19 and MC25</p

Cellular and embryonic localisation of CL-L1.

<p>A. Immunostaining of ATDC5 cells with the golgi marker 58K and CL-L1. CL-L1 shows localisation with golgi apparatus (white arrow). B. Laminin and CL-L1 coimmunolocalisation. Laminin shows partial cellular immunolocalisiton with CL-L1 around the golgi area (arrows). Scale bar 50 μm C. Laminin and CL-K1 coimmunolocalisation. CL-K1 staining shows a very strong golgi localisation with partial cytoplasmatic laminin colocalisation. D. CL-L1 immunohistochemistry of a 18.5 days postfertilisation mouse embryo. CL-L1 is expressed in the liver (long arrow) and submucosal patal region (short arrows). E. Immunofluorescence showing co-localisation of CL-L1 and Laminin in E13.5 mouse embryos sections. CL-L1 is expressed in the basal membrane of the ephithelium in the palate shelf of the maxilla (arrows). In contrast Laminin expression is present all around the ephitelium membrane. A faint but clear CL-L1 expression is also observed in the cytoplasm of the epithelium and in the mesenchyme of the palate. PSM; rostral extremity of right palatal shelf of maxilla. Scale bar 100 μm.</p

New <i>COLEC11</i> and <i>MASP1/3</i> mutations in 3MC patient cohort

<p>New <i>COLEC11</i> and <i>MASP1/3</i> mutations in 3MC patient cohort</p

Clinical findings of individuals homozygous for <i>HYAL2</i> mutations

<p>Clinical findings of individuals homozygous for <i>HYAL2</i> mutations</p

Pedigrees, clinical features of individuals homozygous for <i>HYAL2</i> mutation and identified <i>HYAL2</i> mutations, expression of wild type (WT), K148R-HYAL2 and P250L-HYAL2.

<p>(A) Pedigree diagrams and facial phenotype of individuals (Amish Family 1: XII:7; XII:9; XII:12 and Saudi Family 2: VI:2) with HYAL2 deficiency. Note the craniofacial similarities including frontal bossing, hypertelorism, widened nasal bridge, flattened broad nasal tip and cupped ears/overfolding of the superior helices. Consent for publication of these photographs was obtained (B) Electropherograms showing the identified c.443A>G & c.749C>T mutations and conservation of protein sequence across species.(C) Expression of wild type (WT), K148R and P250L-HYAL2. Western blots were performed on lysates prepared from MEFs deficient in HYAL2 that were transfected with empty vector, pCMV6-HYAL2, pCMV6-HYAL2K148R, or pCMV6-HYAL2P250L. An arrow indicates HYAL2. HYAL2 levels shown in the graph were quantified by imaging the chemiluminescent signal using a BioRad ChemiDoc. The columns represent the average level (x 10⁶ light units) of HYAL2 ± SEM (n = 4). Significance was determined using the student’s T test. *** indicates p<0.0001.</p

Identification of regulatory deletions telomeric to <i>PAX6</i>.

<p>Regulatory deletions telomeric to <i>PAX6</i> were identified in individual 1514 (chr11:30,874,642–31,654,833), individual 753 (chr11:30,967,000–31,704,000), individual 555 (chr11:31,108,579–31,649–842), individual 2014 (chr11:31,234,395–31,751,815) and individual 659 (chr11:31,379,000–31,708,000). The schematic diagram shows how the ‘critical region’ (delimited by grey dotted lines) required for <i>PAX6</i> transcriptional activation was delineated by combining our data with published deletions with known coordinates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153757#pone.0153757.ref055" target="_blank">55</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153757#pone.0153757.ref067" target="_blank">67</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153757#pone.0153757.ref068" target="_blank">68</a>]. <i>PAX6</i> regulatory deletions from the present study are shown by red blocks. Genes transcribed on the forward strand are in blue and those transcribed on the reverse strand are in green, also indicated by arrows. Genomic coordinates are based on the Human Genome Assembly hg18.</p

Details of the clinical diagnoses and genetic pathology identified in individuals in this study.

<p>Details of the clinical diagnoses and genetic pathology identified in individuals in this study.</p

Mutation analysis of the <i>FOXC1</i> locus.

<p><b>(A)</b> Genome-wide array CGH identified two deletions encompassing the <i>FOXC1</i> gene in individuals 1449 (chr6:1,543,591–1,675,085) and 1246 (chr6:1,543,591–1,675,085). <b>(B)</b> Direct sequencing of the <i>FOXC1</i> coding region identified a heterozygous substitution in individual 1839 (c.235C>A, p.(Pro79Thr)) and another in individual 1634 (c.302T>C, p.(Leu101Pro)). <i>FOXC1</i> mutation screening in unaffected parents of both patients showed that the mutations had occurred <i>de novo</i>. The locations of both mutations within the fork-head domain of the FOXC1 protein are indicated by vertical arrows. Genes transcribed on the forward strand are in blue and those transcribed on the reverse strand are in green, also indicated by arrows. Genomic coordinates are based on the Human Genome Assembly hg18. The genomic sequence identifier for <i>FOXC1</i> is NG_009368.</p