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.

Initiation of metformin in early pregnancy results in fetal bioaccumulation, growth restriction, and renal dysmorphology in a primate model.

BACKGROUND: In recent years, pragmatic metformin use in pregnancy has stretched to include prediabetes mellitus, type 2 diabetes mellitus, gestational diabetes mellitus, and (most recently) preeclampsia. However, with its expanded use, concerns of unintended harm have been raised. OBJECTIVE: This study developed an experimental primate model and applied ultrahigh performance liquid chromatography coupled to triple-quadrupole mass spectrometry for direct quantitation of maternal and fetal tissue metformin levels with detailed fetal biometry and histopathology. STUDY DESIGN: Within 30 days of confirmed conception (defined as early pregnancy), 13 time-bred (timed-mated breeding) Rhesus dams with pregnancies designated for fetal necropsy were initiated on twice-daily human dose-equivalent 10 mg/kg metformin or vehicle control. Pregnant dams were maintained as pairs and fed either a control chow or 36% fat Western-style diet. Metformin or placebo vehicle control was delivered in various treats while the animals were separated via a slide. A cesarean delivery was performed at gestational day 145, and amniotic fluid and blood were collected, and the fetus and placenta were delivered. The fetus was immediately necropsied by trained primate center personnel. All fetal organs were dissected, measured, sectioned, and processed per clinical standards. Fluid and tissue metformin levels were assayed using validated ultrahigh performance liquid chromatography coupled to triple-quadrupole mass spectrometry in selected reaction monitoring against standard curves. RESULTS: Among 13 pregnancies at gestational day 145 with fetal necropsy, 1 dam and its fetal tissues had detectable metformin levels despite being allocated to the vehicle control group (>1 µmol metformin/kg maternal weight or fetal or placental tissue), whereas a second fetus allocated to the vehicle control group had severe fetal growth restriction (birthweight of 248.32 g [<1%]) and was suspected of having a fetal congenital condition. After excluding these 2 fetal pregnancies from further analyses, 11 fetuses from dams initiated on either vehicle control (n=4: 3 female and 1 male fetuses) or 10 mg/kg metformin (n=7: 5 female and 2 male fetuses) were available for analyses. Among dams initiated on metformin at gestational day 30 (regardless of maternal diet), significant bioaccumulation within the fetal kidney (0.78-6.06 µmol/kg; mean of 2.48 µmol/kg), liver (0.16-0.73 µmol/kg; mean of 0.38 µmol/kg), fetal gut (0.28-1.22 µmol/kg; mean of 0.70 µmol/kg), amniotic fluid (0.43-3.33 µmol/L; mean of 1.88 µmol/L), placenta (0.16-1.00 µmol/kg; mean of 0.50 µmol/kg), fetal serum (0.00-0.66 µmol/L; mean of 0.23 µmol/L), and fetal urine (4.10-174.10 µmol/L; mean of 38.5 µmol/L) was observed, with fetal levels near biomolar equivalent to maternal levels (maternal serum: 0.18-0.86 µmol/L [mean of 0.46 µmol/L]; maternal urine: 42.60-254.00 µmol/L [mean of 149.30 µmol/L]). Western-style diet feeding neither accelerated nor reduced metformin bioaccumulations in maternal or fetal serum, urine, amniotic fluid, placenta, or fetal tissues. In these 11 animals, fetal bioaccumulation of metformin was associated with less fetal skeletal muscle (57% lower cross-sectional area of gastrocnemius) and decreased liver, heart, and retroperitoneal fat masses (P<.05), collectively driving lower delivery weight (P<.0001) without changing the crown-rump length. Sagittal sections of fetal kidneys demonstrated delayed maturation, with disorganized glomerular generations and increased cortical thickness. This renal dysmorphology was not accompanied by structural or functional changes indicative of renal insufficiency. CONCLUSION: Our study demonstrates fetal bioaccumulation of metformin with associated fetal growth restriction and renal dysmorphology after maternal initiation of the drug within 30 days of conception in primates. Given these results and the prevalence of metformin use during pregnancy, additional investigation of any potential immediate and enduring effects of prenatal metformin use is warranted.

Secondary caries adjacent to bulk fill resin composite restorations lined with fluoride agents.

PURPOSE: To evaluate the potential secondary caries reduction of bulk-fill resin composite restorations treated with different fluoride (F)-based agents prior to restoration placement. METHODS: 25 upper extracted molars received Class V cavities on both buccal and lingual surfaces with an enamel margin and root surface margin. The teeth were randomly assigned into five groups with five teeth per group (n=10). The treatment groups were: control group, with no treatment; the other groups received treatment using different F-based agents prior to bonding. All teeth were restored using a selective etch bonding technique and bulk fill resin composite. All teeth received 10,000 thermal cycles followed by immersion in demineralizing solution to produce artificial caries-like lesions. Polarized light microscope evaluation was performed on longitudinal sections (30 enamel lesions and 30 root surface lesions per group). Lesion depth and wall lesions for both enamel and root surface adjacent to the restorations were evaluated. RESULTS: Both lesion depths and frequency of wall lesions in the enamel and root surfaces in all treatment groups were significantly reduced compared with the control group. Among the different F-based agents, no significant difference was seen on the enamel or root surface lesion depth, showing a similar presence of enamel and root cavosurface wall lesions. CLINICAL SIGNIFICANCE: F-based agent treatment of adjacent enamel and root surfaces prior to placement of restorative materials provided resistance to the development of secondary caries during an in vitro caries model.

Genetic diagnosis and renal biopsy findings in the setting of a renal genetics clinic.

As genetic testing becomes more available, its utilization as an early diagnostic tool in nephrology is more common. The objective of the study is to examine diagnostic agreement between the renal biopsy findings and genetic diagnoses. A retrospective study was conducted in February 2022. A total of 28 patients had both genetic diagnosis and histologic results (n = 1 nephrectomy, n = 27 biopsy). We collected clinical, renal biopsy findings, and genetic information. The relationship between the histologic findings and the genetic diagnoses was classified as: concordant, nonspecific, and discordant. A total of 15 males and 13 females were included (mean age = 9.6 years). Clinical suspicion of Alport syndrome was the most common reason for referral (n = 11, 39.3%), followed by nephrotic syndrome (n = 8, 28.5%), "other" (n = 6, 21.4%), cystic kidney disease (n = 1, 3.6%), isolated hematuria (n = 1, 3.6%), and non-nephrotic proteinuria (n = 1, 3.6%). The overall concordance rate between renal histologic and genetic diagnoses was 71.4% (20/28), nonspecific biopsy results were observed in 17.9% (5/28), and discordant results were observed in 10.7% (3/28). All patients referred for suspected Alport Syndrome had pathogenic/likely pathogenic variants in one of the COL4A genes. Two cases of Lowe syndrome and one of PAX2-associated nephropathy had discordant histology findings. Agreement between renal histologic findings and genetic results varies based on the reason for referral. There was a complete agreement for patients referred for Alport Syndrome; However, there were examples that renal biopsy showed secondary findings that were not specifically associated with the underlying genetic results.

Genomic analysis and preclinical xenograft model development identify potential therapeutic targets for MYOD1-mutant soft-tissue sarcoma of childhood.

The myogenic differentiation 1 gene (MYOD1) p.L122R somatic mutation was first discovered in a subset of clinically aggressive embryonal rhabdomyosarcomas and has since been described in both pediatric and adult spindle cell/sclerosing rhabdomyosarcomas. Relatively little is known about the clinical, molecular, and histopathological features of these tumors in children. In order to further characterize the genomic and clinical features of pediatric MYOD1-mutant sarcomas, we evaluated a cohort of soft-tissue sarcoma patients treated at Texas Children's Hospital. Tumor DNA was subjected to next-generation panel sequencing and/or Sanger sequencing of the MYOD1 hotspot mutation. The MYOD1 p.L122R mutation was identified in six tumors, with a variant allele fraction greater than 0.8 in three cases, suggestive of loss of heterozygosity. One sclerosing rhabdomyosarcoma lacking the MYOD1 hotspot mutation was observed to have a MYOD1 copy number gain, also with evidence of loss of heterozygosity. Cancer gene panel sequencing revealed potentially targetable alterations in six of seven (86%) patients with MYOD1 alterations, including four patients with an alteration in the PI3K-AKT pathway: two hotspot PIK3CA mutations and deletions in PTEN and TSC2. On histopathologic review, MYOD1-altered tumors exhibited spindle and/or round cells and varying degrees of hyaline sclerosis. At last follow-up, six patients had died of disease and the seventh progressed early and was subsequently lost to follow-up. Both pre- and post-therapy patient-derived xenograft models were generated from one patient's tumor. These models were confirmed to harbor the MYOD1 and PIK3CA mutations seen in the primary tumor and were shown to be sensitive to PI3K/mTOR inhibition in vitro and in vivo. In conclusion, this study adds to recent reports describing the clinicopathologic and genomic features of MYOD1-altered soft-tissue sarcomas in children, including dismal prognosis and potential molecular targets for therapy. The novel preclinical models developed will facilitate further biological and preclinical study of this rare and aggressive tumor. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Adenosine A2B receptor: A pathogenic factor and a therapeutic target for sensorineural hearing loss.

Over 466 million people worldwide are diagnosed with hearing loss (HL). About 90% of HL cases are sensorineural HL (SNHL) with treatments limited to hearing aids and cochlear implants with no FDA-approved drugs. Intriguingly, ADA-deficient patients have been reported to have bilateral SNHL, however, its underlying cellular and molecular basis remain unknown. We report that Ada-/- mice, phenocopying ADA-deficient humans, displayed SNHL. Ada-/- mice cochlea with elevated adenosine caused substantial nerve fiber demyelination and mild hair cell loss. ADA enzyme therapy in these mice normalized cochlear adenosine levels, attenuated SNHL, and prevented demyelination. Additionally, ADA enzyme therapy rescued SNHL by restoring nerve fiber structure in Ada-/- mice post two-week drug withdrawal. Moreover, elevated cochlear adenosine in untreated mice was associated with enhanced Adora2b gene expression. Preclinically, ADORA2B-specific antagonist treatment in Ada-/- mice significantly improved HL, nerve fiber density, and myelin compaction. We also provided genetic evidence that ADORA2B is detrimental for age-related SNHL by impairing cochlear myelination in WT aged mice. Overall, understanding purinergic molecular signaling in SNHL in Ada-/- mice allows us to further discover that ADORA2B is also a pathogenic factor underlying aged-related SNHL by impairing cochlear myelination and lowering cochlear adenosine levels or blocking ADORA2B signaling are effective therapies for SNHL.

Pulmonary Histopathology Findings in Patients With STAT3 Gain of Function Syndrome.

INTRODUCTION AND AIM: Multiorgan autoimmunity and interstitial lung disease (ILD) are reported in patients with STAT3 GOF syndrome. RESULTS: We present lung histopathology findings in 3 such children, two of whom underwent wedge biopsies with adequate diagnostic material. Wedge biopsies showed interstitial cellular expansion with linear and nodular aggregates of CD8 positive T lymphocytes, plasma cells, and histiocytes; consistent with lymphocytic interstitial pneumonia pattern (LIP). CD4+ T cells and CD20+ B cells were present but infrequent in the interstitium. FOXP3 cells ranged from 0-5%. Focal interstitial and intraalveolar histiocytes were also seen. Neutrophils and eosinophils were rare/absent. Non-occlusive peribronchial lymphoid aggregates showed equal T and B cells; likely reactive in nature. Pulmonary vessels appeared normal without vasculitis or hypertensive change. There was no interstitial or subepithelial fibrosis or organizing pneumonia. Interlobular septa and visceral pleura were unremarkable. CONCLUSION: Children with multi-system autoimmune disorders with ILD should be investigated for STAT3 GOF syndrome. Lung wedge biopsies are more informative than transbronchial biopsies, if a tissue sampling is indicated. CD8 dominant T cell inflammation seems to be a key driver of ILD. Although interstitial fibrosis was not seen in our small sample, longer follow up is needed to understand the natural history.

Guide for community leaders to meet the challenges of personal preparation in the event of a disaster.

OBJECTIVE: In the aftermath of fires which swept through a regional community in 2013, community leaders were thrust, unprepared, into the disaster recovery arena. The objective of this research was to investigate the subsequent lived experience of these community leaders and, based on this information, develop a guide to meet the challenges for their personal preparation in the context of disaster. DESIGN: Ethical approval for the overarching Community Connections project was provided by Charles Sturt University (H2014073). The project design was informed by an interpretivist paradigm and the methodology embraced participatory action research and thus engaged community members and leaders as research partners. This paper reports on the community leader component of the overarching project. SETTING: Blue Mountains, New South Wales, Australia. PARTICIPANTS: There were 7 interview participants in both 2014 and 2018; 5 participated in both years. Participants were either managers of a local non-government organisation, peak body, school, emergency service or large relief organisation with a local presence. MAIN OUTCOME MEASURE: The development of a guide for the personal preparation of community leaders. RESULTS: The stress of community leaders escalated after the disaster, resulting in a debilitating blurring of professional and personal boundaries, heightened demand on personal knowledge, networking relationships and communication strategies. CONCLUSION: The guide is practical and far reaching; the researchers could not locate anything similar to guide community leaders in their personal planning and preparation for work in disaster recovery.

Generation of patient-derived tumor xenografts from percutaneous tumor biopsies in children with bone sarcomas.

One of the limitations of performing percutaneous biopsies in patients with bone sarcomas is the small amount of tumor that can be obtained for research purposes. Here, we describe our experience developing patient-derived tumor xenografts (PDXs) using percutaneous tumor biopsies in children with bone sarcomas. We generated 14 bone sarcoma PDXs from percutaneous tumor biopsies. We also developed eight bone sarcoma PDXs from surgical resection of primary bone tumors and pulmonary metastases. A multidisciplinary team approach was critical to establish an accurate diagnosis and to provide adequate tumor samples for PDX generation.

Neuroblastoma of the Bone and Bone Marrow Without an Apparent Primary Site: Report of 4 Cases With Long-term Follow-up.

Children with neuroblastoma rarely present with metastatic disease without identifiable primary tumors. We describe the clinical and histopathologic characteristics of 4 patients aged 1, 7, 7, and 11 years with neuroblastoma involving bone or bone marrow without an apparent primary site. One patient presented with a periorbital bone lesion, 1 presented with a distal femoral lesion, and 2 presented with diffuse bone marrow involvement. All tumors were negative for MYCN amplification. All patients were alive without evidence of disease 5 years after completion of multimodality therapy. Patients with neuroblastoma of the bone and bone marrow without an apparent primary site may constitute a unique group characterized by older age at diagnosis, nonamplified MYCN tumors, and good response to treatment.

Assessing the impact of vulnerability on perceptions of social cohesion in the context of community resilience to disaster in the Blue Mountains.

OBJECTIVE: To assess the impact of network communications, community participation and elements of vulnerability on the perception of social cohesiveness in the Blue Mountains local government area (Blue Mountains LGA). DESIGN: A questionnaire was administered to residents of the Blue Mountains LGA. Econometric analysis of the resulting data was undertaken. SETTING: Blue Mountains LGA, Australia. PARTICIPANTS: One thousand one hundred and three residents of the Blue Mountains LGA responded to the questionnaire. MAIN OUTCOME MEASURE(S): The responses enabled the construction of variables measuring individual perceptions of community cohesiveness, their network communications and community participation. Demographic data and data on the vulnerabilities of individuals were also collected. RESULTS: The data were used in an econometric model which identified that network communications and community participation impacted positively on perceptions of social cohesiveness while vulnerability factors had a negative impact. CONCLUSIONS: Remedial action to build community cohesiveness and network communications can be expected to have a positive impact on social cohesiveness. In developing strategies to build community cohesiveness and network communication, particular care needs to be taken to ensure the inclusion of those members of society who are regarded as the most vulnerable.

The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function.

The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1(-/-) muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1(-/-) muscle. Moreover, muscle function was significantly impaired in Rbfox1(-/-) muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology.

The prognostic significance of circulating serum amyloid A and CXC chemokine ligand 4 in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most common pediatric bone cancer.  Despite advances in treatment regimens, the survival rate remains 60-70%.  There is an urgent need to identify prognostic biomarkers, so that targeted therapies can be developed to improve the outcome. PROCEDURE: Our laboratory has previously identified that circulating serum amyloid A (SAA) and CXC chemokine ligand 4 (CXCL4) are upregulated in patients with OS.  In this study, we tested if they could be used as prognostic biomarkers.  We used enzyme-linked immunosorbent assays to measure their concentrations in serum samples (n = 233) and immunohistochemistry to examine their expressions in primary tumors (n = 37).  Prognostic significance of the serum concentrations and tumor expressions of the biomarkers was then evaluated. RESULTS: Patients with "high SAA" and "low CXCL4" circulating levels at diagnosis significantly correlated with a worse outcome (HR = 1.68, P = 0.014), which was independent of the metastatic status.  These patients also exhibited a significantly higher rate of poor histologic response to chemotherapy.  Furthermore, low tumor expression of CXCL4 correlated with poor survival (HR = 3.57, P = 0.005). CONCLUSIONS: Our results demonstrate that circulating SAA and CXCL4 may serve as prognostic biomarkers in OS.  Targeting CXCL4 has been reported, suggesting that it may be exploited as a therapeutic target in OS.

IL-33 is required for disposal of unnecessary cells during ovarian atresia through regulation of autophagy and macrophage migration.

Physiological processes such as ovarian follicle atresia generate large amounts of unnecessary cells or tissue detritus, which needs to be disposed of rapidly. IL-33 is a member of the IL-1 cytokine gene family. Constitutive expression of IL-33 in a wide range of tissues has hinted at its role beyond immune defense. We have previously reported a close correlation between IL-33 expression patterns and ovarian atresia. In this study, we demonstrated that IL-33 is required for disposal of degenerative tissue during ovarian atresia using Il33(-/-) mice. Deletion of the Il33 gene impaired normal disposal of atretic follicles, resulting in massive accumulations of tissue wastes abundant with aging-related catabolic wastes such as lipofuscin. Accumulation of tissue wastes in Il33(-/-) mice, in turn, accelerated ovarian aging and functional decline. Thus, their reproductive life span was shortened to two thirds of that for Il33(+/-) littermates. IL-33 orchestrated disposal mechanism through regulation of autophagy in degenerating tissues and macrophage migration into the tissues. Our study provides direct evidence supporting an expanded role of IL-33 in tissue integrity and aging through regulating disposal of unnecessary tissues or cells.

Post-transplant Recurrent Bile Salt Export Pump Disease: A Form of Antibody-mediated Graft Dysfunction and Utilization of C4d.

Recurrent bile salt export pump (rBSEP) disease has been reported in progressive familial intrahepatic cholestasis type 2 (PFIC2) patients following liver transplantation (LT) and is often refractory to standard anti-cellular rejection immunosuppressants. The mechanism of rBSEP disease is proposed to be a form of type II hypersensitivity reaction with de novo anti-BSEP antibodies blocking the function of allograft BSEP. Utilization of C4d has not been evaluated in rBSEP. We describe a girl with 3 episodes of rBSEP with severe pruritus at 8.9, 10.3, and 11.0 years post-LT, respectively. Patient's serum reacted with normal liver canaliculi by indirect immunofluorescence (IF), whereas patient's liver showed canalicular immunoglobulin G deposition. The histologic features of all 3 liver biopsies recapitulate PFIC2 with cholestatic giant cell hepatitis. Canalicular BSEP expression was not detected in areas of feathery degeneration by immunohistochemistry, but was retained in morphologically normal liver. By direct IF, C4d showed diffuse sinusoidal staining in the third biopsy. Patient responded well to rituximab with or without intravenous immunoglobulin with subsiding symptoms and normalization of serum bile acid levels. In conclusion, rBSEP disease should be considered in the differential diagnosis when evaluating for rejection in a PFIC2 patient post-LT presenting with pruritus. A portion of liver core may be snap frozen in OCT medium for possible direct IF for C4d, that can serve as a surrogate marker for complement activation and antibody-mediated graft dysfunction.

Myofibroma in Infancy and Childhood.

Myofibromas are rare fibroblastic-myofibroblastic tumors in children. Their biological behavior is unpredictable and spontaneous regressions have been described. This is a retrospective review of clinical characteristics, treatment, and outcome of children diagnosed with myofibroma between 1999 and 2013 at Texas Children's Hospital. The median age at diagnosis of 42 patients was 37 months. Approximately two thirds of the patients were male. The median length of follow-up was 50.5 months (range, 0 to 165 mo). Thirty-eight patients (90%) had solitary lesions; 19 (50%) in the head and neck, 10 (26%) in the limbs, and 9 (24%) in the trunk. Twelve patients underwent a complete surgical resection. Of the 30 patients with positive margins, only 1 had tumor progression. Two patients had multicentric involvement, and 2 patients had generalized disease with visceral involvement. One patient with generalized disease and a progressive maxillary sinus mass was treated with vinblastine and methotrexate chemotherapy followed by complete surgical resection. All patients were alive at last follow-up. Myofibromas of childhood demonstrate clinical variability, and may spontaneously regress. Positive surgical margins do not adversely affect outcome. The rare patient with progressive unresectable disease may benefit from chemotherapy.

Neuroendocrine Tumor of the Appendix in Children.

Neuroendocrine tumor (NET) of the appendix is the most common gastrointestinal epithelial tumor in children. The utility of serum markers or the indication for hemicolectomy has not been established in children. In 45 children diagnosed with appendiceal NET, 89% NETs were incidentally found following appendectomy performed for suspected acute appendicitis. The median age was 12 years, and 56% patients were female. Postoperative somatostatin scan (n=5), serum chromogranin A (n=4), and urine 5-HIAA (n=9) were all within normal limits. Pathology slides of 35 patients showed mesoappendiceal invasion in 29% patients, and vascular invasion in 6% patients. Seven patients (16%) underwent hemicolectomy for invasion of mesoappendix (n=5), tumor near the resection margin (n=1), and tumor size 1.5 cm with vascular invasion (n=1). Only 2 hemicolectomy specimens showed disease: one in the appendiceal stump and the other as a micrometastasis in a mesenteric lymph node. There were no recurrences and all patients were alive and without evidence of disease at last follow-up. Pediatric appendiceal NET tends to have a benign clinical course with excellent prognosis. In the absence of carcinoid syndrome, postoperative scans and serum biomarkers do not seem to be useful. With completely resected tumors, the indication for hemicolectomy is unclear.

Hemolytic uremic syndrome as the presenting manifestation of WT1 mutation and Denys-Drash syndrome: a case report.

BACKGROUND: Hemolytic uremic syndrome (HUS) can occur as a primary process due to mutations in complement genes or secondary to another underlying disease. HUS sometimes occurs in the setting of glomerular diseases, and it has been described in association with Denys-Drash syndrome (DDS), which is characterized by the triad of abnormal genitourinary development; a pathognomonic glomerulopathy, diffuse mesangial sclerosis; and the development of Wilms tumor. CASE PRESENTATION: We report the case of a 46, XX female infant who presented with HUS and biopsy-proven thrombotic microangiopathy. Next generation sequencing of genes with known mutations causative of atypical HUS found that she was homozygous for the Complement Factor H H3 haplotype and heterozygous for a variant of unknown significance in the DGKE gene. Whole exome sequencing identified a de novo heterozygous WT1 c.1384C > T; p.R394W mutation, which is classically associated with Denys-Drash syndrome (DDS). At the time of bilateral nephrectomy five months after her initial biopsy, she had diffuse mesangial sclerosis, typical of Denys-Drash syndrome, without evidence of thrombotic microangiopathy. CONCLUSION: This unique case highlights HUS as a rare but important manifestation of WT1 mutation and provides new insight into the genetics underlying this association.

Differential effects of collagen prolyl 3-hydroxylation on skeletal tissues.

Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A) ). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity versus complete ablation of the prolyl 3-hydroxylation complex.

Connective tissue alterations in Fkbp10-/- mice.

Osteogenesis imperfecta (OI) is an inherited brittle bone disorder characterized by bone fragility and low bone mass. Loss of function mutations in FK506-binding protein 10 (FKBP10), encoding the FKBP65 protein, result in recessive OI and Bruck syndrome, of which the latter is additionally characterized by joint contractures. FKBP65 is thought to act as a collagen chaperone, but it is unknown how loss of FKBP65 affects collagen synthesis and extracellular matrix formation. We evaluated the developmental and postnatal expression of Fkbp10 and analyzed the consequences of its generalized loss of function. Fkbp10 is expressed at low levels in E13.5 mouse embryos, particularly in skeletal tissues, and steadily increases through E17.5 with expression in not only skeletal tissues, but also in visceral tissues. Postnatally, expression is limited to developing bone and ligaments. In contrast to humans, with complete loss of function mutations, Fkbp10(-/-) mice do not survive birth, and embryos present with growth delay and tissue fragility. Type I calvarial collagen isolated from these mice showed reduced stable crosslink formation at telopeptide lysines. Furthermore, Fkbp10(-/-) mouse embryonic fibroblasts show retention of procollagen in the cell layer and associated dilated endoplasmic reticulum. These data suggest a requirement for FKBP65 function during embryonic connective tissue development in mice, but the restricted expression postnatally in bone, ligaments and tendons correlates with the bone fragility and contracture phenotype in humans.