BMS1 Is Mutated in Aplasia Cutis Congenita

Aplasia cutis congenita (ACC) manifests with localized skin defects at birth of unknown cause, mostly affecting the scalp vertex. Here, genome-wide linkage analysis and exome sequencing was used to identify the causative mutation in autosomal dominant ACC. A heterozygous Arg-to-His missense mutation (p.R930H) in the ribosomal GTPase BMS1 is identified in ACC that is associated with a delay in 18S rRNA maturation, consistent with a role of BMS1 in processing of pre-rRNAs of the small ribosomal subunit. Mutations that affect ribosomal function can result in a cell cycle defect and ACC skin fibroblasts with the BMS1 p.R930H mutation show a reduced cell proliferation rate due to a p21-mediated G1/S phase transition delay. Unbiased comparative global transcript and proteomic analyses of ACC fibroblasts with this mutation confirm a central role of increased p21 levels for the ACC phenotype, which are associated with downregulation of heterogenous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich splicing factors (SRSFs). Functional enrichment analysis of the proteomic data confirmed a defect in RNA post-transcriptional modification as the top-ranked cellular process altered in ACC fibroblasts. The data provide a novel link between BMS1, the cell cycle, and skin morphogenesis

Increased VEGF‐A promotes multiple distinct aging diseases of the eye through shared pathomechanisms

Abstract While increased VEGF‐A has been associated with neovascular age‐related macular degeneration (AMD), it is not known whether VEGF‐A may also promote other age‐related eye diseases. Here, we show that an increase in VEGF‐A is sufficient to cause multiple distinct common aging diseases of the eye, including cataracts and both neovascular and non‐exudative AMD‐like pathologies. In the lens, increased VEGF‐A induces age‐related opacifications that are associated with ERK hyperactivation, increased oxidative damage, and higher expression of the NLRP3 inflammasome effector cytokine IL‐1β. Similarly, increased VEGF‐A induces oxidative stress and IL‐1β expression also in the retinal pigment epithelium (RPE). Targeting NLRP3 inflammasome components or Il1r1 strongly inhibited not only VEGF‐A‐induced cataract formation, but also both neovascular and non‐exudative AMD‐like pathologies. Moreover, increased VEGF‐A expression specifically in the RPE was sufficient to cause choroidal neovascularization (CNV) as in neovascular AMD, which could be inhibited by RPE‐specific inactivation of Flk1, while Tlr2 inactivation strongly reduced CNV. These findings suggest a shared pathogenic role of VEGF‐A‐induced and NLRP3 inflammasome‐mediated IL‐1β activation for multiple distinct ocular aging diseases

Mutations in KCTD1 Cause Scalp-Ear-Nipple Syndrome

Scalp-ear-nipple (SEN) syndrome is a rare, autosomal-dominant disorder characterized by cutis aplasia of the scalp; minor anomalies of the external ears, digits, and nails; and malformations of the breast. We used linkage analysis and exome sequencing of a multiplex family affected by SEN syndrome to identify potassium-channel tetramerization-domain-containing 1 (KCTD1) mutations that cause SEN syndrome. Evaluation of a total of ten families affected by SEN syndrome revealed KCTD1 missense mutations in each family tested. All of the mutations occurred in a KCTD1 region encoding a highly conserved bric-a-brac, tram track, and broad complex (BTB) domain that is required for transcriptional repressor activity. KCTD1 inhibits the transactivation of the transcription factor AP-2 alpha (TFAP2A) via its BTB domain, and mutations in TFAP2A cause cutis aplasia in individuals with branchiooculofacial syndrome (BOFS), suggesting a potential overlap in the pathogenesis of SEN syndrome and BOFS. the identification of KCTD1 mutations in SEN syndrome reveals a role for this BTB-domain-containing transcriptional repressor during ectodermal development.National Institutes of Health National Human Genome Research InstituteLife Sciences Discovery FundWashington Research FoundationMassachusetts Gen Hosp, Cutaneous Biol Res Ctr, Charlestown, MA 02129 USAUniv Washington, Dept Pediat, Seattle, WA 98195 USAUniv Washington, Dept Genome Sci, Seattle, WA 98195 USAUniv Western Sydney Macarthur, Sch Med, Campbelltown, NSW 2560, AustraliaGenet Learning Disabil Serv, Newcastle, NSW 2298, AustraliaJohns Hopkins Univ, Sch Med, McKusick Nathans Inst Genet Med, Baltimore, MD 21205 USAUniversidade Federal de São Paulo, Dept Morphol & Genet, Clin Genet Ctr, BR-04021001 São Paulo, BrazilPontificia Univ Catolica Parana, Dept Internal Med, BR-1155 Curitiba, Parana, BrazilWestern Gen Hosp, South East Scotland Clin Genet Serv, Edinburgh EH4 2XU, Midlothian, ScotlandUniv Florence, Dept Genet & Mol Med, I-50132 Florence, ItalyHop Necker Enfants Malad, Dept Genet, INSERM, U781, F-75015 Paris, FranceUniv Paris Descartes Sorbonne Paris Cite, Inst Imagine, F-75015 Paris, FranceHop Cote Nacre, CHU Caen, Serv Genet, F-14033 Caen 9, FranceUniv Connecticut, Ctr Hlth, Dept Reconstruct Sci, Farmington, CT 06030 USABoston Childrens Hosp, Dept Plast & Oral Surg, Boston, MA 02115 USATreuman Katz Ctr Pediat Bioeth, Seattle Childrens Res Inst, Seattle, WA 98101 USAUniversidade Federal de São Paulo, Dept Morphol & Genet, Clin Genet Ctr, BR-04021001 São Paulo, BrazilNational Institutes of Health National Human Genome Research Institute: 1U54HG006493National Institutes of Health National Human Genome Research Institute: 1RC2HG005608National Institutes of Health National Human Genome Research Institute: 5RO1HG004316Life Sciences Discovery Fund: 2065508Life Sciences Discovery Fund: 0905001Web of Scienc

NLRP3 Inflammasome Blockade Inhibits VEGF-A-Induced Age-Related Macular Degeneration

The NLRP3 inflammasome is activated in age-related macular degeneration (AMD), but it remains unknown whether its activation contributes to AMD pathologies. VEGF-A is increased in neovascular (“wet”) AMD, but it is not known whether it plays a role in inflammasome activation, whether an increase of VEGF-A by itself is sufficient to cause neovascular AMD and whether it can contribute to nonexudative (“dry”) AMD that often co-occurs with the neovascular form. Here, it is shown that an increase in VEGF-A results in NLRP3 inflammasome activation and is sufficient to cause both forms of AMD pathologies. Targeting NLRP3 or the inflammasome effector cytokine IL-1β inhibits but does not prevent VEGF-A-induced AMD pathologies, whereas targeting IL-18 promotes AMD. Thus, increased VEGF-A provides a unifying pathomechanism for both forms of AMD; combining therapeutic inhibition of both VEGF-A and IL-1β or the NLRP3 inflammasome is therefore likely to suppress both forms of AMD

ACC fibroblasts with mutated BMS1 show a delay in 18S pre-rRNA processing.

<p>a. Pre-rRNA processing pathways in human cells (adapted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003573#pgen.1003573-Rouquette1" target="_blank">[33]</a>). Two pathways coexist depending on the order of cleavage in the 5′-ETS (sites A₀ and A₁) and ITS1 (A₂). Nomenclature of the cleavage sites follows Hadjiolova et al. (1993) and Rouquette et al. (2005) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003573#pgen.1003573-Rouquette1" target="_blank">[33]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003573#pgen.1003573-Hadjiolova1" target="_blank">[34]</a>. b. Pulse-chase labeling of pre-rRNAs with L-[<i>methyl</i>-³H]methionine shows that ACC^{BMS1(p.R930H)} fibroblasts (ACC) form all pre-rRNA species compared to control fibroblasts (WT), but retain increased 45S and 30S pre-rRNAs (seen at 180 minutes after pulse-labeling). Relative values for bands normalized to 28S rRNA band are shown (fold-change compared to WT). c. Magnification of 45S pre-rRNAs 180 minutes after pulse labeling shows retained 45S pre-rRNA in ACC^{BMS1(p.R930H)} fibroblasts compared to control fibroblasts (WT). d. Northern blot analysis of pre-rRNA processing using a radioactive ITS-1 probe. RNA from ACC fibroblasts was used carrying the BMS1 p.R930H mutation, as well as from control fibroblasts (WT). The Northern blot shown on the left side for the WT and ACC cells was exposed overnight, while the image on the right side shows the same blot after a 4 hour exposure. In addition, RNA from fibroblasts stably transfected with an inducible BMS1 shRNA vector was used, after shRNA-mediated knockdown of BMS1 transcripts was induced by doxycycline (doxy) treatment. Ethidiumbromide stained gels prior to blotting (bottom) confirm equal loading of RNA. Quantitation of band intensity ratios expressed as relative values (fold-change compared to WT or untreated cells). e. Doxycyline-inducible knockdown of BMS1 by shRNA lentivirus (shBMS1). Doxycycline treatment for 48 hours results in ∼60% reduction of BMS1 transcript levels. * P-value<0.05.</p

ACC fibroblasts with mutated BMS1 show a G1/S phase cell cycle transition delay and reduced cell proliferation rate.

<p>a. Primary ACC fibroblasts show a significantly reduced proportion of cells in S-phase compared to control fibroblasts (WT), consistent with a delay in G1/S phase transition in mutant cells. PI staining and FACS-based analysis. * P-value<0.05 for cells in G1 and S phase. b. Cell proliferation rate is reduced in ACC fibroblasts. * P-value<0.05 for values between d4 to d7. c. In vitro scratch assay of fibroblasts on fibronectin-coated dishes show increased cell migration rate in ACC fibroblasts (assayed at 14 hours after scratch). Images taken with a 2.5× objective. * P-value<0.05. Graph shows area not repopulated with fibroblasts.</p

An increase in p21 levels in ACC skin fibroblasts.

<p>a. Microarray gene expression profiling experiments (experimental triplicates for ACC vs control fibroblasts) show an upregulation of p21 mRNA levels and a downregulation of SRSF3 mRNA levels in ACC fibroblasts. b. These findings were confirmed by semiquantitative RT-PCR. *P-values<0.05. c. Overexpression of mutant BMS1p.R930H in wild-type fibroblasts phenocopies ACC fibroblasts in showing increased p21 and decreased SRSF3 transcript levels. *P-values<0.05. d. Western blotting experiments show increased p21 protein levels in ACC fibroblasts compared to control fibroblasts, while p53 protein levels are not significantly changed. Relative values for bands normalized to loading control are shown.</p

Global proteomic analysis in ACC skin fibroblasts.

<p>a. Comparative quantitative proteomic analysis with iTRAQ labeling experiments. Upregulated proteins in ACC fibroblasts are shown in the upper part of the figure and downregulated proteins in the lower part of the figure. The columns show values for the following: Peptides (95%): number of peptides identified with at least 95% confidence; raw p-value; false discovery rate (FDR), fold change of WT vs ACC mean values. b. Network analysis (Ingenuity) identified the most significantly altered networks to include top functions for “RNA-posttranscriptional modification”, “protein synthesis” and “cell cycle” (Fisher exact test –lg p-values indicated). c. Western blotting experiments confirm downregulation of hnRNPA2B1 in ACC fibroblasts compared to control fibroblasts. Relative values for bands normalized to loading control are shown.</p

A mutation in the ribosome biogenesis GTPase BMS1 causes autosomal-dominant ACC.

<p>a. Representative images of individuals with ACC in a five-generation pedigree with autosomal dominant inheritance pattern and full penetrance. (Left image: 1 year old with hypertrophic scar at site of congenital ACC of the scalp; Middle image: father of 1 year old of left image with scar at site of congenital ACC; Right image: daughter of this father on day 1 after birth with localized erosion on vertex of scalp (indicated with arrow in pedigree; this member was born after the linkage study was completed and carried the disease allele as well)). Family members' DNA used for genome-wide SNP genotyping indicted with black stars; additional family members' DNA included for confirmation of linkage with microsatellite markers indicated in red stars. b. Histogram of multi-point LOD scores along chromosome 10 shows a single chromosomal region with LOD scores >2 on chromosome 10q11. c. A heterozygous missense mutation resulting in a G>A nucleotide change in the BMS1 gene was identified in all affected members with ACC in this family (c.2789G>A). d. The c.2789G>A mutation results in a Arg-to-His amino acid change (p.R930H) of a conserved Arg residue in BMS1. e. The p.R930H amino acid change affects an Arg residue within the putative C-terminal GAP domain of BMS1. Representative image of the domains of BMS1 with its GTPase domain at the N-terminus (adapted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003573#pgen.1003573-Karbstein1" target="_blank">[9]</a>). f. Murine embryonic sections showing the site of skin formation at the vertex area of the scalp at E13.5 (f. and g.). BMS1 expression is seen in all cells, including the proliferative epidermis (C-terminus of BMS1 in red, pH3b green). C-term indicates labeling with the monoclonal anti-BMS1 antibody that recognizes the C-terminus of BMS1. pH3b indicates staining for the proliferation marker phospho-Histone 3B (Ser10) (pH3b, green). g. Co-localization of BMS1 C-terminal (C-term red) and N-terminal domains (N-term green) at E13.5 of embryonic murine scalp at E13.5. Nuclei are labeled with DAPI. All images are acquired with a 20× objective.</p