The mammalian Arg/N-end rule pathway and missense mutations in human <i>UBR1</i> that underlie specific cases of the Johanson-Blizzard syndrome (JBS).

<p>(A) The mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. ‘Primary’, ‘secondary’ and ‘tertiary’ denote mechanistically distinct subsets of destabilizing N-terminal residues (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s1" target="_blank">Introduction</a>). C* denotes oxidized Cys, either Cys-sulfinate or Cys-sulfonate. MetAPs, Met-aminopeptidases. (B) Single-residue mutations in the UBR1 proteins of JBS patients #1 and #2. The positions of mutant residues are indicated both for the original mutations in human UBR1 and for their mimics in <i>S. cerevisiae</i>. (C) Same as in B but the mutation in UBR1 of patient #3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s2" target="_blank">Results</a>).</p

Functional activity of yeast Ubr1 mimics of missense JBS-UBR1 mutants.

<p>(A) Relative enzymatic activity of βgal in extracts from <i>S. cerevisiae</i> JD55 (<i>ubr1Δ</i>) that expressed His-βgal or Tyr-βgal, and also carried an empty vector, or an otherwise identical plasmid expressing wild-type <i>S. cerevisiae</i> Ubr1, or (separately) its three missense mutants Ubr1^V146L, Ubr1^H160R, or Ubr1^Q1224E. The activity of βgal was measured in triplicates, with standard deviations shown. (B) Relative levels of induction of the peptide transporter Ptr2 were assayed by measuring the activity of a plasmid-borne <i>lacZ</i> (βgal-encoding) reporter that was expressed from the P<i>_PTR2</i> promoter in <i>ubr1Δ S. cerevisiae</i> that carried either an empty vector or otherwise identical plasmids that expressed either wild-type Ubr1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Xia1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a> or its indicated mutants. Cells were grown to A₆₀₀ of ∼0.8 in SC(-Ura, -Leu) medium at 30°C, followed by measurements, in triplicate, of βgal activity in cell extracts, with standard deviations shown. (C) The lysine-requiring JD55 (<i>ubr1Δ</i>) <i>S. cerevisiae</i> strain was grown on plates containing 110 µM lysine (Lys) or 66 µM Lys-Ala dipeptide as the sole source of Lys in the medium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang3" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a>. JD52 (<i>ubr1Δ</i>) cells carried a vector plasmid or otherwise identical plasmids expressing wild-type Ubr1 or its missense mutants Ubr1^H160R, Ubr1^V146L and Ubr1^Q1224E. Cells were grown to A₆₀₀ of ∼1 in SC(-Leu) medium at 30°C, washed in sterile water, serially diluted 5-fold, spotted on SC(-Leu, -Lys) plates containing 110 µM Lys or 66 µM Lys-Ala, and incubated at 30°C for 3 days. (D) Cell extracts (equal total protein levels) from experiments described in panels A and B were subjected to SDS-PAGE, followed by immunoblotting with affinity-purified anti-Ubr1 antibody (upper panel) and anti-tubulin antibody (a loading control; lower panel). Asterisk indicates a protein that crossreacts with anti-Ubr1 antibody. (E) Extracts from human lymphocytes (equal amounts of total protein) were subjected to SDS-PAGE, followed by immunoblotting with antibody to human UBR1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s4" target="_blank">Materials and Methods</a>). Lane 1, wild-type lymphocytes. Lane 2, same as lane 1 but from lymphocytes of patient #2 (see the main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g002" target="_blank">2</a>). Lane 3, same as lane 1 but with lymphocytes from patient #3. Lane 4, same as lane 1, but with lymphocytes from a JBS patient with a homozygous nonsense mutation in <i>UBR1</i>, previously shown to have no detectable UBR1 (null UBR1 control) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Varshavsky3" target="_blank">[17]</a>. Lane 5, same as a lane 1.</p

<i>S. cerevisiae</i> Ubr1 N-recognin.

<p>(A) A diagram of the 225 kDa <i>S. cerevisiae</i> Ubr1. The indicated evolutionarily conserved regions of Ubr1 are the UBR box, the BRR (basic residues-rich) domain, the Cys/His-rich RING-H2 domain, and the AI (<u>a</u>uto<u>in</u>hibitory) domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Tasaki1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Xie1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang3" target="_blank">[33]</a>. Three missense mutations in patients #1-3 of the present work are indicated as well (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B, C</a>). (B) Ribbon diagram of the <i>S. cerevisiae</i> UBR domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Choi1" target="_blank">[48]</a> in a complex with RLGES, the N-terminal region of the separase-produced fragment of Scc1, a subunit of cohesin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Rao1" target="_blank">[75]</a>. The bound RLGES peptide is shown as a stick model, with carbon atoms colored yellow. Several residues are marked with a black sphere and numbered to facilitate the tracing of the polypeptide chain. The names of residues of the RLGES peptide are in red, with the letter ‘s’ (substrate) appended to their position numbers. Side-chains of residues in the UBR domain that are present near JBS mutations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B, C</a>) are shown in a stick form, with carbon atoms colored green. Three coordinated zinc ions of the UBR domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Choi1" target="_blank">[48]</a> are shown as red spheres. (C) Close-up view of the UBR region near the V146L mutation (patient #1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B</a>). In panel B, this region of UBR is boxed and labeled as ‘C’. The residues of UBR that accommodate the position-2 Leu residue (‘Leu2s’) of the RLGES peptide substrate are shown and labeled. The van der Waals sphere of the mutant Leu residue, in the UBR1^V146L mutant, is shown as purple dots. (D) Close-up view of the UBR region near the H160R mutation (patient #2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B</a>). In panel B, this region of UBR is boxed and labeled as ‘D’. The residues of UBR coordinating Zn3 atom are shown and labeled. The van der Waals sphere of the mutant Arg residue, in the UBR1^H160R mutant, is shown as purple dots. The views in (C) and (D) are oriented to maximize visibility of mutation-proximal residues.</p

Clinical features in JBS patients.

a<p>Ref. 1;</p>b<p>Ref. 6 and unpublished data.</p

Additional file 2: of Novel promoters and coding first exons in DLG2 linked to developmental disorders and intellectual disability

Knowledge-driven analysis results. (XLSX 13 kb

Epidemiology of septo-optic dysplasia with focus on prevalence and maternal age - A EUROCAT study.

Septo-optic nerve dysplasia is a rare congenital anomaly with optic nerve hypoplasia, pituitary hormone deficiencies and midline developmental defects of the brain. The clinical findings are visual impairment, hypopituitarism and developmental delays. The aim of this study was to report prevalence, associated anomalies, maternal age and other epidemiological factors from a large European population based network of congenital anomaly registries (EUROCAT). Data from 29 full member registries for the years 2005-2014 were included, covering 6.4 million births. There were 99 cases with a diagnosis of septo-optic dysplasia. The prevalence of septo-optic dysplasia in Europe was calculated to lie between 1.9 and 2.5 per 100,000 births after adjusting for potential under-reporting in some registries. The prevalence was highest in babies of mothers aged 20-24 years of age and was significantly higher in UK registries compared with other EUROCAT registries (P = 0.021 in the multilevel model) and the additional risk for younger mothers was significantly greater in the UK compared to the rest of Europe (P = 0.027). The majority of septo-optic dysplasia cases were classified as an isolated cerebral anomaly (N = 76, 77%). Forty percent of diagnoses occurred in fetuses with a prenatal diagnosis. The anomaly may not be visible at birth, which is reflected in that 57% of the postnatal diagnoses occurred over 1 month after birth. This is the first population based study to describe the prevalence of septo-optic dysplasia in Europe. Septo-optic dysplasia shares epidemiological patterns with gastroschisis and this strengthens the hypothesis of vascular disruption being an aetiological factor for septo-optic dysplasia

Prevalence of anomalies from 2003 to 2012, the annual proportional change during this period and the adjusted annual proportional change after excluding outliers for the 17 anomaly subgroups with statistically significant trends identified in Fig 1.

<p>Prevalence of anomalies from 2003 to 2012, the annual proportional change during this period and the adjusted annual proportional change after excluding outliers for the 17 anomaly subgroups with statistically significant trends identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194986#pone.0194986.g001" target="_blank">Fig 1</a>.</p

Prevalence and annual average change in prevalence for congenital cystic adenomatous malformation of lung and oesophageal atresia.

<p>[A] European Prevalence 1981–2012 (95% CI) with trend for 2003–2012 (black line) and trend excluding outliers (red line) [B] Prevalence for 2003–2012: European (99% CI vertical grey line) and registry (95% CI) [C] Annual change in prevalence for 2003–2012: European (black line and 99% CI funnel) and registry (linear trend black dots, non-linear trend open diamonds). Red line is no trend.</p

Prevalence and annual average change in prevalence for maternal infections resulting in malformations.

<p>[A] European Prevalence 1981–2012 (95% CI) with trend for 2003–2012 (black line) and trend excluding outliers (red line) [B] Prevalence for 2003–2012: European (99% CI vertical grey line) and registry (95% CI) [C] Annual change in prevalence for 2003–2012: European (black line and 99% CI funnel) and registry (linear trend black dots, non-linear trend open diamonds). Red line is no trend.</p

Prevalence and annual average change in prevalence for microcephaly and severe congenital heart disease.

<p>[A] European Prevalence 1981–2012 (95% CI) with trend for 2003–2012 (black line) and trend excluding outliers (red line) [B] Prevalence for 2003–2012: European (99% CI vertical grey line) and registry (95% CI) [C] Annual change in prevalence for 2003–2012: European (black line and 99% CI funnel) and registry (linear trend black dots, non-linear trend open diamonds). Red line is no trend. Severe CHD includes single ventricle, hypoplastic left heart, hypoplastic right heart, Ebstein anomaly, tricuspid atresia, pulmonary valve atresia, common arterial truncus, atrioventricular septal defects, aortic valve atresia/stenosis, transposition of great vessels, tetralogy of Fallot, total anomalous pulmonary venous return, and coarctation of aorta.</p