NRP1 haploinsufficiency predisposes to the development of Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart defect. It involves anatomical abnormalities that change the normal flow of blood through the heart resulting in low oxygenation. Although not all of the underlying causes of TOF are completely understood, the disease has been associated with varying genetic etiologies including chromosomal abnormalities and Mendelian disorders, but can also occur as an isolated defect. In this report, we describe a familial case of TOF associated with a 1.8 Mb deletion of chromosome 10p11. Among the three genes in the region one is Neuropilin1 (NRP1), a membrane co-receptor of VEGF that modulates vasculogenesis. Hemizygous levels of NRP1 resulted in a reduced expression at the transcriptional and protein levels in patient-derived cells. Reduction of NRP1 also lead to decreased function of its activity as a co-receptor in intermolecular VEGF signaling. These findings support that diminished levels of NRP1 contribute to the development of TOF, likely through its function in mediating VEGF signal and vasculogenesis

NRP1 haploinsufficiency predisposes to the development of Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart defect. It involves anatomical abnormalities that change the normal flow of blood through the heart resulting in low oxygenation. Although not all of the underlying causes of TOF are completely understood, the disease has been associated with varying genetic etiologies including chromosomal abnormalities and Mendelian disorders, but can also occur as an isolated defect. In this report, we describe a familial case of TOF associated with a 1.8 Mb deletion of chromosome 10p11. Among the three genes in the region one is Neuropilin1 (NRP1), a membrane co-receptor of VEGF that modulates vasculogenesis. Hemizygous levels of NRP1 resulted in a reduced expression at the transcriptional and protein levels in patient-derived cells. Reduction of NRP1 also lead to decreased function of its activity as a co-receptor in intermolecular VEGF signaling. These findings support that diminished levels of NRP1 contribute to the development of TOF, likely through its function in mediating VEGF signal and vasculogenesis

WDR34 Mutations that Cause Short-Rib Polydactyly Syndrome Type III/Severe Asphyxiating Thoracic Dysplasia Reveal a Role for the NF-κB Pathway in Cilia

Short-rib polydactyly (SRP) syndrome type III, or Verma-Naumoff syndrome, is an autosomal-recessive chondrodysplasia characterized by short ribs, a narrow thorax, short long bones, an abnormal acetabulum, and numerous extraskeletal malformations and is lethal in the perinatal period. Presently, mutations in two genes, IFT80 and DYNC2H1, have been identified as being responsible for SRP type III. Via homozygosity mapping in three affected siblings, a locus for the disease was identified on chromosome 9q34.11, and homozygosity for three missense mutations in WDR34 were found in three independent families, as well as compound heterozygosity for mutations in one family. WDR34 encodes a member of the WD repeat protein family with five WD40 domains, which acts as a TAK1-associated suppressor of the IL-1R/TLR3/TLR4-induced NF-κB activation pathway. We showed, through structural modeling, that two of the three mutations altered specific structural domains of WDR34. We found that primary cilia in WDR34 mutant fibroblasts were significantly shorter than normal and had a bulbous tip. This report expands on the pathogenesis of SRP type III and demonstrates that a regulator of the NF-κB activation pathway is involved in the pathogenesis of the skeletal ciliopathies

Clinical and radiographic delineation of Bent Bone Dysplasia‐FGFR2 type or Bent Bone Dysplasia with Distinctive Clavicles and Angel‐shaped Phalanges

Bent Bone Dysplasia-FGFR2 type is a relatively recently described bent bone phenotype with diagnostic clinical, radiographic, and molecular characteristics. Here we report on 11 individuals, including the original four patients plus seven new individuals with three longer-term survivors. The prenatal phenotype included stillbirth, bending of the femora, and a high incidence of polyhydramnios, prematurity, and perinatal death in three of 11 patients in the series. The survivors presented with characteristic radiographic findings that were observed among those with lethality, including bent bones, distinctive (moustache-shaped) small clavicles, angel-shaped metacarpals and phalanges, poor mineralization of the calvarium, and craniosynostosis. Craniofacial abnormalities, hirsutism, hepatic abnormalities, and genitourinary abnormalities were noted as well. Longer-term survivors all needed ventilator support. Heterozygosity for mutations in the gene that encodes Fibroblast Growth Factor Receptor 2 (FGFR2) was identified in the nine individuals with available DNA. Description of these patients expands the prenatal and postnatal findings of Bent Bone Dysplasia-FGFR2 type and adds to the phenotypic spectrum among all FGFR2 disorders. © 2016 Wiley Periodicals, Inc

Bent Bone Dysplasia-FGFR2 type, a Distinct Skeletal Disorder, Has Deficient Canonical FGF Signaling

Fibroblast growth factor receptor 2 (FGFR2) is a crucial regulator of bone formation during embryonic development. Both gain and loss-of-function studies in mice have shown that FGFR2 maintains a critical balance between the proliferation and differentiation of osteoprogenitor cells. We have identified de novo FGFR2 mutations in a sporadically occurring perinatal lethal skeletal dysplasia characterized by poor mineralization of the calvarium, craniosynostosis, dysmorphic facial features, prenatal teeth, hypoplastic pubis and clavicles, osteopenia, and bent long bones. Histological analysis of the long bones revealed that the growth plate contained smaller hypertrophic chondrocytes and a thickened hypercellular periosteum. Four unrelated affected individuals were found to be heterozygous for missense mutations that introduce a polar amino acid into the hydrophobic transmembrane domain of FGFR2. Using diseased chondrocytes and a cell-based assay, we determined that these mutations selectively reduced plasma-membrane levels of FGFR2 and markedly diminished the receptor's responsiveness to extracellular FGF. All together, these clinical and molecular findings are separate from previously characterized FGFR2 disorders and represent a distinct skeletal dysplasia

TGFβ nuclear target RNA expression increased in absence of FLNB <i>in vivo</i>.

<p>(A,B) RT-qPCR analysis results using RNA derived from P1 mouse sternums. The expression levels of the TGFβ nuclear targets <i>CTGF</i> and <i>p21</i>, are both significantly increased in <i>Flnb</i>^{<i>–/–</i>}mouse sternums, N = 6. * = p<0.05, ** = p<0.01, data are represented as mean ± SEM. (C,D) RT-qPCR analysis results using RNA derived from P15 mouse IVDs. The expression levels of the TGFβ nuclear targets, <i>CTGF</i> and <i>p21</i>, are significantly increased in <i>Flnb</i>^{<i>–/–</i>}mouse IVDs, N = 3. *** = p<0.001, **** = p<0.0001, NS = not significant, data are represented as mean ± SEM.</p

TGFβ and BMP Dependent Cell Fate Changes Due to Loss of Filamin B Produces Disc Degeneration and Progressive Vertebral Fusions

<div><p>Spondylocarpotarsal synostosis (SCT) is an autosomal recessive disorder characterized by progressive vertebral fusions and caused by loss of function mutations in <i>Filamin B</i> (<i>FLNB)</i>. FLNB acts as a signaling scaffold by linking the actin cytoskleteon to signal transduction systems, yet the disease mechanisms for SCT remain unclear. Employing a <i>Flnb</i> knockout mouse, we found morphologic and molecular evidence that the intervertebral discs (IVDs) of <i>Flnb</i>^{<i>–/–</i>}mice undergo rapid and progressive degeneration during postnatal development as a result of abnormal cell fate changes in the IVD, particularly the annulus fibrosus (AF). In <i>Flnb</i>^{<i>–/–</i>}mice, the AF cells lose their typical fibroblast-like characteristics and acquire the molecular and phenotypic signature of hypertrophic chondrocytes. This change is characterized by hallmarks of endochondral-like ossification including alterations in collagen matrix, expression of Collagen X, increased apoptosis, and inappropriate ossification of the disc tissue. We show that conversion of the AF cells into chondrocytes is coincident with upregulated TGFβ signaling via Smad2/3 and BMP induced p38 signaling as well as sustained activation of canonical and noncanonical target genes <i>p21</i> and <i>Ctgf</i>. These findings indicate that FLNB is involved in attenuation of TGFβ/BMP signaling and influences AF cell fate. Furthermore, we demonstrate that the IVD disruptions in <i>Flnb</i>^{<i>–/–</i>}mice resemble aging degenerative discs and reveal new insights into the molecular causes of vertebral fusions and disc degeneration.</p></div

TGFβ and BMP pathway components exhibit nuclear localization in <i>Flnb</i>^{<i>–/–</i>}AF.

<p>IHC analysis of P15 mouse disc tissue paraffin sections. (A, A′, E, E′) Whole IVDs shown for orientation. (B, B′, F, F′) DAPI channel only showing nuclear staining. (C, C′, G, G′) Red channel only showing protein localization. (D) P-Smad1,5,8 (red) has a cytoplasmic localization in the <i>Flnb</i>^<i>+/+</i> AF. (D′) P-Smad1,5,8 (red) exhibits a nuclear localization as it co-localizes with the nucleus (blue) In the AF of <i>Flnb</i>^{<i>–/–</i>}mice. (H) P-Smad3 (red) has a cytoplasmic localization in the <i>Flnb</i>^<i>+/+</i> AF. (H′) P-Smad3 (red) exhibits a nuclear localization as it co-localizes with the nucleus (blue) in the AF of <i>Flnb</i>^{<i>–/–</i>}mice. (I-L) Western blot analysis of fractionated protein lysates extracted from cultured primary mouse sternal chondrocytes. (I,J) p-Smad1,5,8 levels remain unchanged in the cytoplasm but are significantly higher in the nuclei of stimulated primary chondrocytes. (K,L) p-Smad3 levels are significantly upregulated in both the cytoplasm and the nucleus without stimulation. (K,L) N = 3. NS = Not Significant, * = p<0.05, ** = p<0.01, data are represented as mean ± SEM.</p

Illustration of tissue morphology change in <i>Flnb</i>^{<i>–/–</i>}IVD.

<p>(A) <i>Flnb</i>^<i>+/+</i> IVD illustration. (B) <i>Flnb</i>^{<i>–/–</i>}IVD illustrating transition of AF to hypertrophic-like state resembling the mineralized endplate. Flanking vertebral bodies shift in position as IVD degenerates resulting in inappropriate spinal curvature. (C) IVD of 5 year old SCT patient. Positions of flanking vertebrae suggest compression of the IVD identical to those observed in <i>Flnb</i>^{<i>–/–</i>}mouse spines.</p

BMP non-canonical pathway activation is increased in the absence of FLNB <i>in vivo</i> and <i>in vitro</i>.

<p>(A-C) Western blot analysis of protein lysates extracted from cultured primary mouse chondrocytes. (A) P-Smad1,5,8 levels remain unchanged in <i>Flnb</i>^{<i>–/–</i>}when compared with <i>Flnb</i>^<i>+/+</i>, N = 6. (B) P-p38 levels are increased endogenously as well as upon BMP-2 (10ng/mL) stimulation in mutant versus <i>Flnb</i>^<i>+/+</i> chondrocytes, N = 3. (C) p-ERK levels are upregulated endogenously but are subsequently decreased in mutant chondrocytes following 30 minutes of BMP-2 stimulation. N = 4. (D) Luciferase assay measuring <i>Msx2</i> promoter activity in primary mouse chondrocytes. <i>Msx2</i> promoter activity is increased in <i>Flnb</i>^{<i>–/–</i>}chondrocytes, N = 3. * = p<0.05, ** = p<0.01, data are represented as mean ± SEM. (E) Western blot analysis of protein lysates extracted from AF tissue of P15 pups. BMP-2 and p-p38 protein expression levels are increased in <i>Flnb</i>^{<i>–/–</i>}IVDs with no change in p-Smad1/5/8 levels, N = 7.</p