COPB2 loss of function causes a coatopathy with osteoporosis and developmental delay.

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.

TRIM44 promotes quiescent multiple myeloma cell occupancy and survival in the osteoblastic niche via HIF-1α stabilization.

Despite progress in the treatment of MM, including the use of high-dose chemotherapy and autologous stem cell transplantation, a considerable proportion of patients are refractory to all therapies. This resistance is related to the molecular genetic heterogeneity in MM cells as well as to the contributions from the BM, which is one of the key determinants of treatment outcome. Our previous studies using fluorescent tracers revealed that MM heterogeneity is correlated with the presence of quiescent stem-like cancer cells, which prefer to reside within the osteoblastic niche of the BM. In this report, we identified a novel protein, tripartite motif containing 44 (TRIM44), which is overexpressed in the osteoblastic niche of the BM, enabling MM cells to compete with HSCs for niche support. TRIM44 expression in MM cells promoted cell quiescence but increased bone destruction in xenograft mice, similar to what is observed in MM patients. TRIM44 functions as a deubiquitinase for hypoxia inducible factor-1α (HIF-1α), which stabilizes HIF-1α expression during hypoxia and normoxia. Stabilized HIF-1α stimulates MM cell growth and survival during hypoxia. Our work is the first report to reveal signaling in quiescent MM cells and the functions of TRIM44.

MicroRNA miR-23a cluster promotes osteocyte differentiation by regulating TGF-ß signalling in osteoblasts.

Osteocytes are the terminally differentiated cell type of the osteoblastic lineage and have important functions in skeletal homeostasis. Although the transcriptional regulation of osteoblast differentiation has been well characterized, the factors that regulate differentiation of osteocytes from mature osteoblasts are poorly understood. Here we show that miR-23a∼27a∼24-2 (miR-23a cluster) promotes osteocyte differentiation. Osteoblast-specific miR-23a cluster gain-of-function mice have low bone mass associated with decreased osteoblast but increased osteocyte numbers. By contrast, loss-of-function transgenic mice overexpressing microRNA decoys for either miR-23a or miR-27a, but not miR24-2, show decreased osteocyte numbers. Moreover, RNA-sequencing analysis shows altered transforming growth factor-ß (TGF-ß) signalling. Prdm16, a negative regulator of the TGF-ß pathway, is directly repressed by miR-27a with concomitant alteration of sclerostin expression, and pharmacological inhibition of TGF-ß rescues the phenotypes observed in the gain-of-function transgenic mice. Taken together, the miR-23a cluster regulates osteocyte differentiation by modulating the TGF-ß signalling pathway through targeting of Prdm16.

RECQL4 Regulates p53 Function In Vivo During Skeletogenesis.

RECQ DNA helicases play critical roles in maintaining genomic stability, but their role in development has been less well studied. Rothmund-Thomson syndrome, RAPADILINO, and Baller-Gerold syndrome are rare genetic disorders caused by mutations in the RECQL4 gene. These patients have significant skeletal developmental abnormalities including radial ray, limb and craniofacial defects. To investigate the role of Recql4 in the developing skeletal system, we generated Recql4 conditional knockout mice targeting the skeletal lineage. Inactivation of Recql4 using the Prx1-Cre transgene led to limb abnormalities and craniosynostosis mimicking the major bone findings in human RECQL4 patients. These Prx1-Cre(+) ;Recql4(fl/fl) mice as well as Col2a1-Cre(+) ;Recql4(fl/fl) mice exhibited growth plate defects and an increased p53 response in affected tissues. Inactivation of Trp53 in these Recql4 mutants resulted in genetic rescue of the skeletal phenotypes, indicating an in vivo interaction between Recql4 and Trp53, and p53 activation as an underlying mechanism for the developmental bone abnormalities in RECQL4 disorders. Our findings show that RECQL4 is critical for skeletal development by modulating p53 activity in vivo.

Gene sequence variability of the three surface proteins of human respiratory syncytial virus (HRSV) in Texas.

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004-2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community.

Phenotypic variability of osteogenesis imperfecta type V caused by an IFITM5 mutation.

In a large cohort of osteogenesis imperfecta type V (OI type V) patients (17 individuals from 12 families), we identified the same mutation in the 5' untranslated region (5'UTR) of the interferon-induced transmembrane protein 5 (IFITM5) gene by whole exome and Sanger sequencing (IFITM5 c.-14C > T) and provide a detailed description of their phenotype. This mutation leads to the creation of a novel start codon adding five residues to IFITM5 and was recently reported in several other OI type V families. The variability of the phenotype was quite large even within families. Whereas some patients presented with the typical calcification of the forearm interosseous membrane, radial head dislocation and hyperplastic callus (HPC) formation following fractures, others had only some of the typical OI type V findings. Thirteen had calcification of interosseous membranes, 14 had radial head dislocations, 10 had HPC, 9 had long bone bowing, 11 could ambulate without assistance, and 1 had mild unilateral mixed hearing loss. The bone mineral density varied greatly, even within families. Our study thus highlights the phenotypic variability of OI type V caused by the IFITM5 mutation.

The KAT6B-related disorders genitopatellar syndrome and Ohdo/SBBYS syndrome have distinct clinical features reflecting distinct molecular mechanisms.

Genitopatellar syndrome (GPS) and Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) have both recently been shown to be caused by distinct mutations in the histone acetyltransferase KAT6B (a.k.a. MYST4/MORF). All variants are de novo dominant mutations that lead to protein truncation. Mutations leading to GPS occur in the proximal portion of the last exon and lead to the expression of a protein without a C-terminal domain. Mutations leading to SBBYSS occur either throughout the gene, leading to nonsense-mediated decay, or more distally in the last exon. Features present only in GPS are contractures, anomalies of the spine, ribs and pelvis, renal cysts, hydronephrosis, and agenesis of the corpus callosum. Features present only in SBBYSS include long thumbs and long great toes and lacrimal duct abnormalities. Several features occur in both, such as intellectual disability, congenital heart defects, and genital and patellar anomalies. We propose that haploinsufficiency or loss of a function mediated by the C-terminal domain causes the common features, whereas gain-of-function activities would explain the features unique to GPS. Further molecular studies and the compilation of mutations in a database for genotype-phenotype correlations (www.LOVD.nl/KAT6B) might help tease out answers to these questions and understand the developmental programs dysregulated by the different truncations.

miRNA-34c regulates Notch signaling during bone development.

During bone homeostasis, osteoblast and osteoclast differentiation is coupled and regulated by multiple signaling pathways and their downstream transcription factors. Here, we show that microRNA 34 (miR-34) is significantly induced by BMP2 during osteoblast differentiation. In vivo, osteoblast-specific gain of miR-34c in mice leads to an age-dependent osteoporosis due to the defective mineralization and proliferation of osteoblasts and increased osteoclastogenesis. In osteoblasts, miR-34c targets multiple components of the Notch signaling pathway, including Notch1, Notch2 and Jag1 in a direct manner, and influences osteoclast differentiation in a non-cell-autonomous fashion. Taken together, our results demonstrate that miR-34c is critical during osteoblastogenesis in part by regulating Notch signaling in bone homeostasis. Furthermore, miR-34c-mediated post-transcriptional regulation of Notch signaling in osteoblasts is one possible mechanism to modulate the proliferative effect of Notch in the committed osteoblast progenitors which may be important in the pathogenesis of osteosarcomas. Therefore, understanding the functional interaction of miR-34 and Notch signaling in normal bone development and in bone cancer could potentially lead to therapies modulating miR-34 signaling.

Protein interactome reveals converging molecular pathways among autism disorders.

To uncover shared pathogenic mechanisms among the highly heterogeneous autism spectrum disorders (ASDs), we developed a protein interaction network that identified hundreds of new interactions among proteins encoded by ASD-associated genes. We discovered unexpectedly high connectivity between SHANK and TSC1, previously implicated in syndromic autism, suggesting that common molecular pathways underlie autistic phenotypes in distinct syndromes. ASD patients were more likely to harbor copy number variations that encompass network genes than were control subjects. We also identified, in patients with idiopathic ASD, three de novo lesions (deletions in 16q23.3 and 15q22 and one duplication in Xq28) that involve three network genes (NECAB2, PKM2, and FLNA). The protein interaction network thus provides a framework for identifying causes of idiopathic autism and for understanding molecular pathways that underpin both syndromic and idiopathic ASDs.

A new method to orient 3-dimensional computed tomography models to the natural head position: a clinical feasibility study.

PURPOSE: The purpose of this study was to evaluate the clinical feasibility of a new method to orient 3-dimensional (3D) computed tomography models to the natural head position (NHP). This method uses a small and inexpensive digital orientation device to record NHP in 3 dimensions. This device consists of a digital orientation sensor attached to the patient via a facebow and an individualized bite jig. The study was designed to answer 2 questions: 1) whether the weight of the new device can negatively influence the NHP and 2) whether the new method is as accurate as the gold standard. PATIENTS AND METHODS: Fifteen patients with craniomaxillofacial deformities were included in the study. Each patient's NHP is recorded 3 times. The first NHP was recorded with a laser scanning method without the presence of the digital orientation device. The second NHP was recorded with the digital orientation device. Simultaneously, the third NHP was also recorded with the laser scanning method. Each recorded NHP measurement was then transferred to the patient's 3D computed tomography facial model, resulting in 3 different orientations for each patient: the orientation generated via the laser scanning method without the presence of the digital orientation sensor and facebow (orientation 1), the orientation generated by use of the laser scanning method with the presence of the digital orientation sensor and facebow (orientation 2), and the orientation generated with the digital orientation device (orientation 3). Comparisons are then made between orientations 1 and 2 and between orientations 2 and 3, respectively. Statistical analyses are performed. RESULTS: The results show that in each pair, the difference (Δ) between the 2 measurements is not statistically significantly different from 0°. In addition, in the first pair, the Bland-Altman lower and upper limits of the Δ between the 2 measurements are within 1.5° in pitch and within a subdegree in roll and yaw. In the second pair, the limits of the Δ in all 3 dimensions are within 0.5°. CONCLUSION: Our technique can accurately record NHP in 3 dimensions and precisely transfer it to a 3D model. In addition, the extra weight of the digital orientation sensor and facebow has minimal influence on the self-balanced NHP establishment.

Pancreatic pseudocyst rupture into the portal vein.

A patient with a pancreatic pseudocyst rupture into the portal vein with a resultant noninfectious systemic inflammatory response syndrome and subsequent portal vein thrombosis diagnosed by computed tomography and ultrasonography is reported. A review of the existing English literature on this rare complication is also provided.

RVU ready? Preparing emergency medicine resident physicians in documentation for an incentive-based work environment.

OBJECTIVES: The emergency medicine (EM) job market is increasingly focused on incentive-based reimbursement, which is largely based on relative value units (RVUs) and is directly related to documentation of patient care. Previous studies have shown a need to improve resident education in documentation. The authors created a focused educational intervention on billing and documentation practices to meet this identified need. The hypothesis of this study was that this educational intervention would result in an increase in RVUs generated by EM resident physicians and the average amount billed per patient. METHODS: The authors used a quasi-experimental study design. An educational intervention included a 1-hour lecture on documentation and billing, biweekly newsletters, and case-specific feedback from the billing department for EM resident physicians. RVUs and charges generated per patient were recorded for all second- and third-year resident physicians for a 3-month period prior to the educational intervention and for a 3-month period following the intervention. Pre- and postintervention data were compared using Student's t-test and repeated-measures analysis of variance, as appropriate. RESULTS: The evaluation and management (E/M) chart levels billed during each phase of the study were significantly different (p < 0.0001). The total number of RVUs generated per hour increased from 3.17 in the first phase to 3.71 in the second phase (p = 0.0001). During the initial 3-month phase, the average amount billed per patient seen by a second- or third-year resident was 282.82 USD, which increased to 301.94 USD in the second phase (p = 0.0004). CONCLUSIONS: The educational intervention positively affected resident documentation resulting in greater RVUs/hour and greater billing performance in the study emergency department (ED).