Aberrant Multiciliogenesis in Idiopathic Pulmonary Fibrosis.

We previously identified a novel molecular subtype of idiopathic pulmonary fibrosis (IPF) defined by increased expression of cilium-associated genes, airway mucin gene MUC5B, and KRT5 marker of basal cell airway progenitors. Here we show the association of MUC5B and cilia gene expression in human IPF airway epithelial cells, providing further rationale for examining the role of cilium genes in the pathogenesis of IPF. We demonstrate increased multiciliogenesis and changes in motile cilia structure of multiciliated cells both in IPF and bleomycin lung fibrosis models. Importantly, conditional deletion of a cilium gene, Ift88 (intraflagellar transport 88), in Krt5 basal cells reduces Krt5 pod formation and lung fibrosis, whereas no changes are observed in Ift88 conditional deletion in club cell progenitors. Our findings indicate that aberrant injury-activated primary ciliogenesis and Hedgehog signaling may play a causative role in Krt5 pod formation, which leads to aberrant multiciliogenesis and lung fibrosis. This implies that modulating cilium gene expression in Krt5 cell progenitors is a potential therapeutic target for IPF.

Pathogenic variants in MRPL44 cause infantile cardiomyopathy due to a mitochondrial translation defect.

Cardiac dysfunction is a common phenotypic manifestation of primary mitochondrial disease with multiple nuclear and mitochondrial DNA pathogenic variants as a cause, including disorders of mitochondrial translation. To date, five patients have been described with pathogenic variants in MRPL44, encoding the ml44 protein which is part of the large subunit of the mitochondrial ribosome (mitoribosome). Three presented as infants with hypertrophic cardiomyopathy, mild lactic acidosis, and easy fatigue and muscle weakness, whereas two presented in adolescence with myopathy and neurological symptoms. We describe two infants who presented with cardiomyopathy from the neonatal period, failure to thrive, hypoglycemia and in one infant lactic acidosis. A decompensation of the cardiac function in the first year resulted in demise. Exome sequencing identified compound heterozygous variants in the MRPL44 gene including the known pathogenic variant c.467 T > G and two novel pathogenic variants. We document a combined respiratory chain enzyme deficiency with emphasis on complex I and IV, affecting heart muscle tissue more than skeletal muscle or fibroblasts. We show this to be caused by reduced mitochondrial DNA encoded protein synthesis affecting all subunits, and resulting in dysfunction of complex I and IV assembly. The degree of oxidative phosphorylation dysfunction correlated with the impairment of mitochondrial protein synthesis due to different pathogenic variants. These functional studies allow for improved understanding of the pathogenesis of MRPL44-associated mitochondrial disorder.

TNF-α-driven inflammation and mitochondrial dysfunction define the platelet hyperreactivity of aging.

Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.

Use of electron microscopy when screening liver biopsies from neonates and infants: experience from a single tertiary children's hospital (1991-2017).

Background: Although the role of electron microscopy is diminishing in several areas of adult pathology, it remains an essential tool for the study of pediatric liver biopsies.Methods: Clinical charts, histologic slides and EM materials of native liver biopsies from patients <1 year old (1991-2017) were reviewed.Results: 677 biopsies were performed on 353 males and 324 females. This study presents the concrete numbers for both the indications and the diseases, and describes the role of EM. EM was performed on 24.7% of liver biopsies and demonstrated key pathologic findings in 10 cases (6%), which led to the appropriate biochemical and/or genetic testing to confirm the diagnoses. The cases included five cases of glycogen storage disease with characteristic findings with cytoplasmic glycogen accumulation, two cases of mitochondrial disorder with pleomorphic mitochondria with crystalloid inclusions and one case each of Niemann-Pick Disease with abundant myelinosomes, Alpha-1 antitrypsin deficiency with deposits in the endoplasmic reticulum and infantile Refsum disease with trilamellar inclusions and lack of peroxisomes. In this study, we describe the detailed histologic and EM findings of each case .Conclusion: EM played an important screening and diagnostic role in the challenging cases and was also used to rule out detectable pathologic conditions.

Mutations in the accessory subunit NDUFB10 result in isolated complex I deficiency and illustrate the critical role of intermembrane space import for complex I holoenzyme assembly.

An infant presented with fatal infantile lactic acidosis and cardiomyopathy, and was found to have profoundly decreased activity of respiratory chain complex I in muscle, heart and liver. Exome sequencing revealed compound heterozygous mutations in NDUFB10, which encodes an accessory subunit located within the PD part of complex I. One mutation resulted in a premature stop codon and absent protein, while the second mutation replaced the highly conserved cysteine 107 with a serine residue. Protein expression of NDUFB10 was decreased in muscle and heart, and less so in the liver and fibroblasts, resulting in the perturbed assembly of the holoenzyme at the 830 kDa stage. NDUFB10 was identified together with three other complex I subunits as a substrate of the intermembrane space oxidoreductase CHCHD4 (also known as Mia40). We found that during its mitochondrial import and maturation NDUFB10 transiently interacts with CHCHD4 and acquires disulfide bonds. The mutation of cysteine residue 107 in NDUFB10 impaired oxidation and efficient mitochondrial accumulation of the protein and resulted in degradation of non-imported precursors. Our findings indicate that mutations in NDUFB10 are a novel cause of complex I deficiency associated with a late stage assembly defect and emphasize the role of intermembrane space proteins for the efficient assembly of complex I.

Histologic and ultrastructural features in early and advanced phases of Zellweger spectrum disorder (infantile Refsum disease).

Zellweger spectrum disorders (ZSD) are rare autosomal recessive inherited metabolic disorders and include severe (Zellweger syndrome) and milder phenotypes [neonatal adrenoleukodystrophy and infantile Refsum disease (IRD)]. ZSD are characterized by impaired peroxisomal functions and lack of peroxisomes detected by electron microscopy (EM). ZSD are caused by mutations in any of the 14 PEX genes. Patients with ZSD commonly demonstrate nonspecific hepatic symptoms within the first year, often without clinical suspicion of ZSD. Thus, recognition of pathologic findings in the liver is critical for the early diagnosis. We herein demonstrate the histologic and ultrastructural features in liver biopsies in the early and advanced phases from a 16-year-old male with IRD. The initial biopsy at 5 months of age showed a lack of peroxisomes by EM, and this finding played a critical role in the early diagnosis. In contrast, the second biopsy at 14 years of age, after long-term diet therapy, demonstrated significant disease progression with near-cirrhotic liver. In addition to lack of peroxisomes, EM revealed abundant trilamellar inclusions within large angulated lysosomes in many of the hepatocytes and Kupffer cells. Mitochondrial abnormalities were identified only in the second biopsy and were mainly identified in damaged cells; thus they were likely nonspecific secondary changes. This is the first report demonstrating histological and ultrastructural features of liver biopsies in the early and advanced phases from a child with ZSD. Trilamellar inclusions are considered to be an ultrastructural hallmark of ZSD, but they may not be apparent in the early phases.

Renal diseases associated with hematuria in children and adolescents: a brief tutorial.

The detection of microscopic hematuria in a child's urine prompts evaluation for renal and urinary bladder causes. Microscopic hematuria identified during a routine physical examination by the pediatrician is much more common than macroscopic hematuria. Persistent microscopic hematuria is particularly worrisome and may require a percutaneous needle core kidney biopsy to determine whether the etiology is secondary to glomerular disease, tubulointerstitial disease, urinary tract infection, urinary tract structural abnormalities, medications, or toxins. This paper reviews the epidemiology, pathologic features, pathogenesis, treatment, and outcome of familial hematuria (Alport syndrome [hereditary nephritis]), thin basement membrane nephropathy), IgA nephropathy, Henoch-Schönlein purpura, and acute postinfectious glomerulonephritis.

Ultrastructural features of NUT midline carcinoma.

Midline carcinomas associated with the nuclear protein in testis (NUT) gene rearrangement are rare, aggressive tumors that have been diagnosed most commonly in the head, neck, mediastinum, and upper aerodigestive tract. The ultrastructural features associated with this tumor have thus far received only brief comment and have never been illustrated. The authors provide a more extensive description and illustrate the electron microscopic findings in a typical case of NUT midline carcinoma, confirmed by cytogenetic and fluorescence in situ hybridization studies. This tumor was composed of cells displaying large, irregularly shaped nuclei with prominent compact nucleoli and abundant cytoplasm containing prominent bundles of tonofilaments, occasional clusters of pleomorphic granules, small numbers of lipid inclusions, and rare glycogen deposits. The cells exhibited stubby microvillous projections, were intermittently enveloped by basal lamina, and were interjoined by numerous well-formed desmosomal-type junctions and occasional junctional complexes. The authors propose that this constellation of ultrastructural features can prove helpful in discriminating NUT midline carcinoma from similar appearing entities.

Papillary tumor of the pineal region: ultrastructural study of a case.

Papillary tumor of the pineal region (PTPR) is a recently classified neuroepithelial tumor for which there has been little comprehensive ultrastructural study. Here, we describe the radiographic, intraoperative, histologic, immunohistochemical, and in-depth ultrastructural findings in a case of PTPR. This study corroborates that PTPR has concomitant ependymal, neuroendocrine, and secretory features, and details novel ultrastructural as well as immunohistochemical features that further this argument. Discrepancies with prior descriptions of PTPR are described, as these differences may reflect phenotypic variability in this rare tumor, and the ultrastructural features that relate to the putative ependymal origin of the entity are emphasized.

Loss of FOCAD, operating via the SKI messenger RNA surveillance pathway, causes a pediatric syndrome with liver cirrhosis.

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient's primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

Glycogen storage diseases: a brief review and update on clinical features, genetic abnormalities, pathologic features, and treatment.

Glycogen storage diseases (GSD) affect primarily the liver, skeletal muscle, heart, and sometimes the central nervous system and the kidneys. These unique diseases are quite varied in age of onset of symptoms, morbidity, and mortality. Glycogen storage diseases are classified according to their individual enzyme deficiency. Each of these enzymes regulates synthesis or degradation of glycogen. Interestingly, there is great phenotypic variation and variable clinical courses even when a specific enzyme is altered by mutation. Depending on the specific mutation in an enzyme, a GSD patient may have a favorable or unfavorable prognosis. With neonatal or infantile forms, some GSDs lead to death within the first year of life, whereas other glycogen storage diseases are relatively asymptomatic or may cause only exercise intolerance. The paper provides a brief review and update of glycogen storage diseases, with respect to clinical features, genetic abnormalities, pathologic features, and treatment.

Plexiform fibrohistiocytic tumor: ultrastructural studies may aid in discrimination from cellular neurothekeoma.

Plexiform fibrohistiocytic tumor is a low-grade soft tissue malignancy that can at times be difficult to differentiate from the less biologically aggressive cellular neurothekeoma. The two entities, which may display identical clinical and histological features, cannot be distinguished by immunohistochemical or molecular diagnostic means. Electron microscopy may enable the accurate identification of problematic examples and thus aid in resolving these occasionally occurring diagnostic dilemmas. To illustrate typical variations in the ultrastructural appearance of plexiform fibrohistiocytic tumor, the authors present two diagnostically noncontroversial examples, and to demonstrate the potential diagnostic utility of electron microscopy in this setting, they present an example of plexiform fibrohistiocytic tumor that could not otherwise have been distinguished from cellular neurothekeoma.

Neonatal Onset Interstitial Lung Disease as a Primary Presenting Manifestation of Mucopolysaccharidosis Type I.

We describe two cases of neonatal onset interstitial lung disease eventually diagnosed as mucopolysaccharidosis type I (MPS I). In both cases, evaluation led to lung biopsy, pathology review, and identification of glycogen deposition. Pulmonary interstitial glycogenosis (PIG) was considered as a clinical diagnosis in case one; however, further review of electron microscopy (EM) was more consistent with MPS I rather than PIG. Both cases were confirmed to have MPS I by enzyme and molecular analysis. Neonatal interstitial lung disease is an atypical presentation for MPS I which is likely under-recognized. Diagnosis through clinical guidelines and a multidisciplinary approach had a major impact on patient management. The diagnosis of MPS I prompted timely initiation of enzyme replacement therapy (ERT) and the patients ultimately underwent hematopoietic stem cell transplantation (HSCT) to improve symptomatic outcomes. In addition to treatment, immediate precautionary recommendations were made to avoid potentially catastrophic outcomes associated with cervical instability. These cases add to the clinical spectrum of MPS I in the newborn period. They further illustrate the difficulties in early recognition of the disease, and importance of a definitive diagnosis of MPS I in infants with interstitial lung disease.

Renal medullary carcinoma: ultrastructural studies may benefit diagnosis.

Renal medullary carcinoma is a recently described highly aggressive malignancy that in most instances exhibits a constellation of clinical and light microscopic features sufficiently distinctive to enable a quick and confident diagnosis. Presented are three examples where, because of unusual elements in the clinical presentation, electron microscopic examination proved beneficial in establishing the diagnosis.

Marijuana medusa: The many pulmonary faces of marijuana inhalation in adolescent males.

Marijuana use has risen dramatically over the past decade. Over this same time period, pediatric hospitals have seen an increase in presentation of adolescents with acute respiratory symptoms after recent marijuana inhalation. We report a case series of three adolescent males with significant findings of bilateral pulmonary nodules and ground glass opacities on chest imaging associated with recent marijuana inhalation. Lung biopsies in two of the three patients confirmed silica-induced pneumoconiosis. The third patient was diagnosed with acute hypersensitivity pneumonitis without lung biopsy. Improvement in clinical symptoms and lung function testing were noted in two of three patients after marijuana inhalation cessation. This case series highlights the variety of severe pulmonary presentations in adolescents following recent marijuana inhalation. Future studies are required to assess whether these presenting pulmonary complications are from direct marijuana exposure or indirect associations with marijuana inhalation injuries.

Filamin C Truncation Mutations Are Associated With Arrhythmogenic Dilated Cardiomyopathy and Changes in the Cell-Cell Adhesion Structures.

OBJECTIVES: The purpose of this study was to assess the phenotype of Filamin C (FLNC) truncating variants in dilated cardiomyopathy (DCM) and understand the mechanism leading to an arrhythmogenic phenotype. BACKGROUND: Mutations in FLNC are known to lead to skeletal myopathies, which may have an associated cardiac component. Recently, the clinical spectrum of FLNC mutations has been recognized to include a cardiac-restricted presentation in the absence of skeletal muscle involvement. METHODS: A population of 319 U.S. and European DCM cardiomyopathy families was evaluated using whole-exome and targeted next-generation sequencing. FLNC truncation probands were identified and evaluated by clinical examination, histology, transmission electron microscopy, and immunohistochemistry. RESULTS: A total of 13 individuals in 7 families (2.2%) were found to harbor 6 different FLNC truncation variants (2 stopgain, 1 frameshift, and 3 splicing). Of the 13 FLNC truncation carriers, 11 (85%) had either ventricular arrhythmias or sudden cardiac death, and 5 (38%) presented with evidence of right ventricular dilation. Pathology analysis of 2 explanted hearts from affected FLNC truncation carriers showed interstitial fibrosis in the right ventricle and epicardial fibrofatty infiltration in the left ventricle. Ultrastructural findings included occasional disarray of Z-discs within the sarcomere. Immunohistochemistry showed normal plakoglobin signal at cell-cell junctions, but decreased signals for desmoplakin and synapse-associated protein 97 in the myocardium and buccal mucosa. CONCLUSIONS: We found FLNC truncating variants, present in 2.2% of DCM families, to be associated with a cardiac-restricted arrhythmogenic DCM phenotype characterized by a high risk of life-threatening ventricular arrhythmias and a pathological cellular phenotype partially overlapping with arrhythmogenic right ventricular cardiomyopathy.

Pathogenic variants in glutamyl-tRNAGln amidotransferase subunits cause a lethal mitochondrial cardiomyopathy disorder.

Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNAGln). mt-tRNAGln is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNAGln and mitochondrial protein translation are deficient in patients' fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. Our study completes a decade of investigations on mitochondrial aminoacylation disorders, starting with DARS2 and ending with the GatCAB complex.

Atypical presentation of neuronal ceroid lipofuscinosis type 8 in a sibling pair and review of the eye findings and neurological features.

PURPOSE: To report atypical presentation of neuronal ceroid lipofuscinoses type 8 (CLN8) to the eye clinic and review clinical features of CLN8. OBSERVATIONS: Detailed eye exam by slit lamp exam, indirect ophthalmoscopy, fundus photography, optical coherence tomography, visual fields and electroretinogram (ERG). Molecular genetic testing using Next Generation Sequencing panel (NGS) and array Comparative Genomic Hybridization (aCGH).The siblings in this study presented to the eye clinic with retinitis pigmentosa and cystoid macular edema, and a history of seizures but no severe neurocognitive deficits or regression. Genetic testing identified a c.200C > T (p.A67V) variant in the CLN8 gene and a deletion encompassing the entire gene. Electron microscopy of lymphocytes revealed fingerprint inclusions in both siblings. CONCLUSIONS: and Importance: Pathogenic variants in CLN8 account for the retinitis pigmentosa and seizures in our patients however, currently, they do not have regression or neurocognitive decline. The presentation of NCL can be very diverse and it is important for ophthalmologists to consider this in the differential diagnosis of retinal disorders with seizures or other neurological features. Molecular genetic testing of multiple genes causing isolated and syndromic eye disorders using NGS panels and aCGH along with additional complementary testing may often be required to arrive at a definitive diagnosis.

Ultrastructural features of eosinophilic oesophagitis: impact of treatment on desmosomes.

AIMS: A growing body of evidence suggests a role for altered epithelial barrier function in the pathophysiology of eosinophilic oesophagitis (EoE), but few have described the epithelial structure during inflammation. The purpose of this study was to define ultrastructural features of active, inactive EoE and control subject's oesophageal epithelia. METHODS: We prospectively enrolled patients undergoing diagnostic upper endoscopy for evaluation of EoE. Mucosal pinch biopsies were obtained from the distal oesophagus and processed for routine histology and electron microscopic assessment. Clinical features of enrolled subjects were analysed and subjects were divided into four groups: normal, gastroesophageal reflux disease (GERD), inactive EoE and active EoE. Representative photomicrographs of the basal and superficial epithelia were reviewed for abnormalities. Desmosomes were quantified on the surface of epithelia three to four prickle-cell layers above the basal layer. RESULTS: Twenty-nine paediatric cases (ages 2-18 years) were enrolled in the study. We observed a significant decrease in the number of desmosomes per cell (DPC) of subjects with active EoE compared with inactive EoE, GERD and normal epithelia. With respect to DPC, no significant differences were found between inactive EoE compared with GERD or normal subjects. Additional ultrastructural features observed included epithelial microplicae and evidence of eosinophil transmigration, degranulation, and sombrero formation. CONCLUSIONS: Consistent with clinical and molecular findings, our ultrastructural data provide support for an altered oesophageal barrier in paediatric cases with active EoE, which may improve following treatment.