Schematic phylogenetic trees of the Smyd proteins.

<p>In all the panels sequences are indicated with abbreviated species name and annotated protein name. Branch lengths and bootstrap values are given as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134106#pone.0134106.g001" target="_blank">Fig 1</a>. (A, B) Phylogenetic trees obtained with the alignment of the metazoan Smyd proteins by the maximum likelihood (A) and neighbor joining (B) methods; the branches corresponding to the four main classes are compressed and the two sequences not included in one of these classes are indicated in grey. (C) Maximum likelihood and neighbor joining (D) phylogenetic trees obtained with the alignment of the extended dataset, which includes the sequences from the metazoans plus unicellular metazoan-related species <i>Capsaspora owczarzaki</i> and <i>Monosiga brevicollis</i>, the plant <i>Arabidopsis thaliana</i> and the yeast <i>Saccharomyces cerevisiae</i>. The non-metazoan species are indicated in black type, and those that are grouped within one of the classes are indicated in the compressed branch with the name preceded by +.</p

Sequence analysis of the confirmed and putative MYND domains.

<p>(A) For the protein sequences indicated on the left, the MYND Zn finger within the interrupted SET domain is compared to the PROSITE signature PS01360 and to the MYND hidden Markov model PFAM signature. For the PROSITE signature, the formula is indicated above with Zn-ligand residues in bold and highlighting the four Zn-ligand pairs. We have highlighted in grey all departures from the signature, including residues that do not match the consensus and stretches of the wrong length. For the PFAM model we indicate whether the sequence is a perfect match (+), a partial match of the right or left portions (+(r) and +(l) respectively), or not a match at all (-, highlighted grey). (B) Scheme of the cross-brace disposition of a MYND-type Zn finger, with the most common Zn-ligand residues depicted in black and Zn ions as empty circles; the numbers indicate the number of aminoacids between Zn-ligand residues according to the PS01360 formula.</p

Transcriptional expression profile of <i>Drosophila</i> and mouse <i>Smyd4</i> class genes.

<p>We show a transcriptional study for <i>Drosophila melanogaster</i> genes <i>CG1868</i> (A-A”‘), <i>CG8378</i> (B-B”‘), <i>CG14122</i> (C-C”‘) and <i>CG7759</i> (D-D”‘) and <i>Mus musculus Smyd4</i> (E-G). For the <i>Drosophila</i> genes we show high throughput data from two consortia, the anatomical expression profile of FlyAtlas (A-D) and the temporal expression profile of modENCODE (A’-D’). For these genes we also determined the expression pattern by i<i>n situ</i> hybridization in embryos (anterior to the right, lateral view except where indicated). We show expression in extended germ band (A”-D”) and in stage 16 embryos (A”‘-D”‘). The ventral nerve cord in B”‘ is not visible as the embryo is slightly tilted. We also determined the expression of <i>Smyd4</i> in a 14.5E mouse embryo section. We show sections of the abdomen (E), dorsal trunk (F) and head (G). Abbreviations are used for the tissues where expression is detected as follows: GP, gut primordium; CP, cephalic primordium of the central nervous system; GT, gut; MS, mesoderm; CNS, central nervous system; SP, spinal cord; DRG, dorsal root ganglia; EN, encephalon.</p

Species included in the phylogenetic study of Smyd family proteins.

<p>Species included in the phylogenetic study of Smyd family proteins.</p

Proposed evolutionary history of <i>Smyd</i> genes in metazoans.

<p>The schematic tree represents the accepted phylogeny for the phyla/subphyla represented in this work. From an original complement of <i>Smyd</i> genes, comprising Smyd3, Smyd4 and Smyd5, we indicate with white boxes the most likely events of gene gain (+) duplication (Dup.) and expansion (Exp.); and in black boxes the events of gene loss (-).</p

Table1_Case report: A third variant in the 5′ UTR of TWIST1 creates a novel upstream translation initiation site in a child with Saethre-Chotzen syndrome.docx

Introduction: Saethre-Chotzen syndrome, a craniosynostosis syndrome characterized by the premature closure of the coronal sutures, dysmorphic facial features and limb anomalies, is caused by haploinsufficiency of TWIST1. Although the majority of variants localize in the coding region of the gene, two variants in the 5′ UTR have been recently reported to generate novel upstream initiation codons.Methods: Skeletal dysplasia Next-generation sequencing (NGS) panel was used for genetic analysis in a patient with bicoronal synostosis, facial dysmorphisms and limb anomalies. The variant pathogenicity was assessed by a luciferase reporter promoter assay.Results: Here, we describe the identification of a third ATG-creating de novo variant, c.-18C>T, in the 5′ UTR of TWIST1 in the patient with a clinical diagnosis of Saethre-Chotzen syndrome. It was predicted to create an out-of-frame new upstream translation initiation codon resulting in a 40 amino acid larger functionally inactive protein. We performed luciferase reporter promoter assays to demonstrate that the variant does indeed reduce translation from the main open reading frame.Conclusion: This is the third variant identified in this region and confirms the introduction of upstream ATGs in the 5′ UTR of TWIST1 as a pathogenic mechanism in Saethre-Chotzen syndrome. This case report shows the necessity for performing functional characterization of variants of unknown significance within national health services.</p