Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain ...elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.
•Autosomal recessive loss-of-function mutations in G6b-B (MPIG6B) cause congenital macrothrombocytopenia with focal myelofibrosis.•G6b-B has orthologous physiological functions in human and mice regulating megakaryocyte and platelet production and function.
Display omitted
Choroid plexus tumors and ciliary body medulloepithelioma are predominantly pediatric neoplasms. Progress in understanding the pathogenesis of these tumors has been hindered by their rarity and lack ...of models that faithfully recapitulate the disease. Here, we find that endogenous Myc proto-oncogene protein is down-regulated in the forebrain neuroepithelium, whose neural plate border domains give rise to the anterior choroid plexus and ciliary body. To uncover the consequences of persistent Myc expression, MYC expression was forced in multipotent neural precursors (nestin-Cre:Myc), which produced fully penetrant models of choroid plexus carcinoma and ciliary body medulloepithelioma. Nestin-mediated MYC expression in the epithelial cells of choroid plexus leads to the regionalized formation of choroid plexus carcinoma in the posterior domain of the lateral ventricle choroid plexus and the fourth ventricle choroid plexus that is accompanied by loss of multiple cilia, up-regulation of protein biosynthetic machinery, and hydrocephalus. Parallel MYC expression in the ciliary body leads also to up-regulation of protein biosynthetic machinery. Additionally, Myc expression in human choroid plexus tumors increases with aggressiveness of disease. Collectively, our findings expose a select vulnerability of the neuroepithelial lineage to postnatal tumorigenesis and provide a new mouse model for investigating the pathogenesis of these rare pediatric neoplasms.
Microvillus inclusion disease (MVID) is a rare congenital disorder that manifests early in infancy as intractable watery diarrhea. The entity is characterized morphologically by a deficient brush ...border and apical cytoplasmic inclusions within absorptive cells (enterocytes) due to misplaced assembly of brush border proteins. The diagnosis is based upon histopathology, special stains, immunohistochemistry (IHC), and ultimately upon electron microscopy. Currently, the periodic acid-Schiff stain (PAS) and CD10 IHC are commonly used as adjuncts, but in addition to brush border structures, they stain a variety of apical cytoplasmic inclusions and organelles, thereby interfering with recognition of microvillus inclusions. Villin is a protein that specifically binds to the actin core bundle of microvilli. We utilized villin IHC in formalin-fixed paraffin-embedded gastrointestinal biopsies from 6 patients with MVID, 5 with celiac disease, and 17 children with normal intestinal biopsies and compared the results with those obtained with CD10 IHC and PAS staining. All MVID cases had confirmatory electron microscopy at the time of diagnosis. Villin immunoreactivity was restricted to the brush border in the control groups. In MVID, villin IHC showed attenuation or loss of the surface brush border and also highlighted the cytoplasmic microvillus inclusions with clarity. In MVID, CD10 IHC and the PAS stain also showed attenuation or loss of the surface brush border, but staining of a variety of cytoplasmic structures largely obscured the microvillus inclusions. In sum, villin IHC is a reliable and superior adjunct in the diagnosis of MVID. Study of additional cases will determine whether villin IHC would obviate the need for electron microscopic confirmation.
Tumor-specific immunohistochemical markers are valuable in the differential diagnosis of malignant small round cell tumors (MSRCTs). The cone-rod homeobox-containing gene (CRX) is a transcription ...factor that is preferentially expressed in retinal photoreceptor cells. It has been shown that the CRX antibody is a good immunohistochemical marker to differentiate retinoblastoma from other intracranial MSRCTs. Outside of the central nervous system, however, the usefulness of CRX immunohistochemistry in establishing a diagnosis of metastatic retinoblastoma is uncertain, as the expression of CRX in primitive neuroectodermal tumor/Ewing sarcoma (PNET/ES), neuroblastoma, and other MSRCTs is unknown. Archival specimens from resections, core biopsies, and bone marrow biopsies of 41 neuroblastomas, 24 PNET/ES, 19 embryonal rhabdomyosarcomas, 17 alveolar rhabdomyosarcomas, 17 Wilms tumors, 14 desmoplastic small round cell tumors, 20 medulloblastomas, 9 pineal tumors, 17 melanocytic tumors (compound and Spitz nevi), and 8 retinoblastomas were immunostained for CRX. All retinoblastomas had strong diffuse nuclear immunoreactivity for CRX; 8 of the 20 medulloblastomas showed strong nuclear immunoreactivity either in occasional clusters of tumor cells or in rare single scattered tumor cells; 3 of the 9 pineal tumors showed strong nuclear immunoreactivity in approximately 40% to 50% of the tumor cells. Neuroblastomas, PNET/ES, embryonal rhabdomyosarcomas, alveolar rhabdomyosarcomas, Wilms tumors, desmoplastic small round cell tumors, and melanocytic tumors were all negative. Scant nonspecific cytoplasmic staining was observed in some tumors, mostly PNET/ES. These findings suggest that CRX is a useful marker to discriminate metastatic retinoblastoma from other, more common, MSRCTs of childhood.
The molecular pathways controlling cerebellar Purkinje cell dendrite formation and maturation are poorly understood. The
Purkinje cell degeneration (
pcd) mutant mouse is characterized by mutations ...in
Nna1, a gene discovered in an axonal regenerative context, but whose actual function in development and disease is unknown. We found abnormal development of Purkinje cell dendrites in postnatal
pcd
Sid
mice and linked this deficit to a deletion mutation in exon 7 of
Nna1. With single cell gene profiling and virus-based gene transfer, we analyzed a molecular pathway downstream to
Nna1 underlying abnormal Purkinje cell dendritogenesis in
pcd
Sid
mice. We discovered that mutant
Nna1 dramatically increases intranuclear localization of lysyl oxidase propeptide, which interferes with NF-κB RelA signaling and microtubule-associated protein regulation of microtubule stability, leading to underdevelopment of Purkinje cell dendrites. These findings provide insight into
Nna1's role in neuronal development and why its absence renders Purkinje cells more vulnerable.
► The pcdSid mutant mouse features a deletion of Nna1 exon 7 that affects postnatal PCs ► Molecules downstream from mutant Nna1 mediate underdevelopment of pcdSid PC dendrites ► Increased transfer of Lox propeptide into pcdSid PC nucleus reduces NF-κB RelA levels ► Reduced NF-κB signaling suppresses PC dendrite development via altered MAP1B and MAP2
We have studied hypertrophic and immediately adjacent pre-hypertrophic chondrocytes at the same stage of histologic development in 7
day old post-natal Balb/C mouse physes and epiphyses. Laser ...capture microdissection (LCM) and GeneChip microarray analysis compared the molecular composition of the two hypertrophic chondrocyte regions. Molecules upregulated in dramatically higher levels in the epiphysis were gremlin (58-fold), epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (25-fold), and frizzled related protein (6.4-fold and 5.7-fold). Molecules upregulated in higher levels in the physis were proline arginine-rich end leucine-rich repeat protein (PRELP) (15.6-fold), pyrophosphatase (inorganic) 1 (10-fold) and hedgehog-interacting protein (7.3-fold). Immunocytochemistry for gremlin confirmed specific localization patterns. This study indicates a critical site-specific role for hypertrophic chondrocytes with different synthesis patterns in separate regions even though they appear structurally the same and are at the same stage of development.
Desmoplastic small round cell tumor (DSRCT) is a rare malignant tumor that generally manifests as abdominal paraserosal masses and affects mainly male adolescents and young adults. When presenting ...within visceral organs, the diagnosis of DSRCT poses significant difficulties.
Four primary renal DSRCT in children diagnosed during a 3-year period are the basis of this report. The medical records and pathologic material were reviewed, including immunohistochemical, ultrastructural, and cytogenetic/molecular studies.
The age at presentation was 6 to 8 years, and all children presented with a left renal mass. The tumors measured 3.7 to 13.4 cm and consisted of nests, cords, or sheets of small undifferentiated cells with foci of necrosis and calcification. Desmoplasia was not seen. Tumor cells were immunopositive for vimentin, WT-1 (monoclonal and polyclonal), desmin, cytokeratin, and epithelial membrane antigen. A distinct paranuclear dotlike pattern was observed with vimentin and desmin. Tumor cells possessed rare or focal immunoreactivity for platelet derived growth factor-A and transforming growth factor-beta3, which have been implicated in the pathogenesis of desmoplasia in DSRCT. The EWS-WT1 t(11;22)(p13;q12) translocation was demonstrated in all 4 tumors by fluorescence in situ hybridization and/or reverse transcription-polymerase chain reaction.
DSRCT should be considered in the differential diagnosis of renal tumors composed of small round cells. Undifferentiated morphology and lack of desmoplasia contribute to the difficulty in its recognition. Ancillary studies such as immunohistochemistry may suggest the diagnosis, but cytogenetic and molecular genetic studies are required for confirmation.
Ewing sarcoma is a tumor of the bone and soft tissue characterized by diffuse cell membrane expression of CD99 (MIC2). Single-site, surgically resectable disease is associated with an excellent ...5-year event-free survival; conversely, patients with distant metastases have a poor prognosis. Noninvasive imaging is the standard approach to identifying sites of metastatic disease. We sought to develop a CD99-targeted imaging agent for staging Ewing sarcoma and other CD99-expressing tumors.
We identified a CD99 antibody with highly specific binding in vitro and labeled this antibody with (64)Cu. Mice with either subcutaneous Ewing sarcoma xenograft tumors or micrometastases were imaged with the (64)Cu-labeled anti-CD99 antibody and these results were compared with conventional MRI and 218Ffluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) imaging.
(64)Cu-labeled anti-CD99 antibody demonstrated high avidity for the CD99-positive subcutaneous tumors, with a high tumor-to-background ratio, greater than that demonstrated with FDG-PET. Micrometastases, measuring 1 to 2 mm on MRI, were not detected with FDG-PET but were readily visualized with the (64)Cu-labeled anti-CD99 antibody. Probe biodistribution studies demonstrated high specificity of the probe for CD99-positive tumors.
(64)Cu-labeled anti-CD99 antibody can detect subcutaneous Ewing sarcoma tumors and metastatic sites with high sensitivity, outperforming FDG-PET in preclinical studies. This targeted radiotracer may have important implications for the diagnosis, surveillance, and treatment of Ewing sarcoma. Similarly, it may impact the management of other CD99 positive tumors.
Primary pulmonary vein stenosis (PVS) is a progressive disorder of infants. Although catheter based intervention and chemotherapy are used to manage the disorder, the benefit of these approaches is ...reduced considerably by restenosis. The nature of the intimal cells causing the occlusive lesions in PVS is poorly understood.
Seven PVS cases were studied with antibodies for smooth muscle actin (SMA), muscle-specific actin (MSA), monoclonal desmin, S100 protein, CD31, CD34, CD45RO, CD68, CD99, Ki-67 (MIB-I), and with antibodies directed against several receptor tyrosine kinases (RTK), including platelet-derived growth factor alpha and beta receptor (PDGFR-α and -β), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor 1 and 2 receptor (VEGFR), and stem cell factor receptor (c-kit).
Lesional cells stained strongly and diffusely with SMA and MSA, but not for macrophage, lymphocyte, endothelial markers, or for Ki-67. RTK expression was strong and diffuse for PDGFR-α and -β, FGFR, and VEGFR-2. Lesional cells stained for VEGF and PDGF β receptor was phosphorylated.
The histologic appearance, and the strong diffuse immunoreactivity for smooth muscle markers, indicates that the intimal lesional cells are myofibroblast-like. Expression of various receptor tyrosine kinases and some ligands suggests an autocrine or paracrine role of these proteins in the pathogenesis of the intimal occlusive lesion in PVS.
Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous ...system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain.
Display omitted
•CHMP1A is required for progenitor proliferation in developing brain•Loss of CHMP1A decreases sonic hedgehog secretion•Loss of CHMP1A decreases intraluminal vesicles within multivesicular bodies•CHMP1A regulates SHH secretion on the extracellular vesicle subtype ART-EV
Extracellular vesicles (EVs) are essential for cell-to-cell communication in developing brain. Coulter et al. show that the human microcephaly gene CHMP1A is required for neuroprogenitor proliferation through regulation of vesicular secretion of the growth factor sonic hedgehog (SHH). CHMP1A specifically impairs SHH secretion on a distinctive EV subtype, ART-EV.
PDF
