Mutations causing premature termination codons discriminate and generate cellular and clinical variability in HHT.

ABSTRACT: For monogenic diseases caused by pathogenic loss-of-function DNA variants, attention focuses on dysregulated gene-specific pathways, usually considering molecular subtypes together within causal genes. To better understand phenotypic variability in hereditary hemorrhagic telangiectasia (HHT), we subcategorized pathogenic DNA variants in ENG/endoglin, ACVRL1/ALK1, and SMAD4 if they generated premature termination codons (PTCs) subject to nonsense-mediated decay. In 3 patient cohorts, a PTC-based classification system explained some previously puzzling hemorrhage variability. In blood outgrowth endothelial cells (BOECs) derived from patients with ACVRL1+/PTC, ENG+/PTC, and SMAD4+/PTC genotypes, PTC-containing RNA transcripts persisted at low levels (8%-23% expected, varying between replicate cultures); genes differentially expressed to Bonferroni P < .05 in HHT+/PTC BOECs clustered significantly only to generic protein terms (isopeptide-bond/ubiquitin-like conjugation) and pulse-chase experiments detected subtle protein maturation differences but no evidence for PTC-truncated protein. BOECs displaying highest PTC persistence were discriminated in unsupervised hierarchical clustering of near-invariant housekeeper genes, with patterns compatible with higher cellular stress in BOECs with >11% PTC persistence. To test directionality, we used a HeLa reporter system to detect induction of activating transcription factor 4 (ATF4), which controls expression of stress-adaptive genes, and showed that ENG Q436X but not ENG R93X directly induced ATF4. AlphaFold accurately modeled relevant ENG domains, with AlphaMissense suggesting that readthrough substitutions would be benign for ENG R93X and other less rare ENG nonsense variants but more damaging for Q436X. We conclude that PTCs should be distinguished from other loss-of-function variants, PTC transcript levels increase in stressed cells, and readthrough proteins and mechanisms provide promising research avenues.

Mutational and phenotypic characterization of hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT.

Narrative Performance Level Assignments at Initial Entrustment and Graduation: Integrating EPAs and Milestones to Improve Learner Assessment.

PURPOSE: To determine which narrative performance level for each general pediatrics entrustable professional activity (EPA) reflects the minimum level clinical competency committees (CCCs) felt should be associated with graduation as well as initial entrustment and compare expected narrative performance levels (ENPLs) for each EPA with actual narrative performance levels (ANPLs) assigned to residents at initial entrustment. METHOD: A series of 5 narratives, corresponding to the 5 milestone performance levels, were developed for each of the 17 general pediatrics EPAs. In academic year (AY) 2015-2016, the CCCs at 22 Association of Pediatric Program Directors Longitudinal Educational Assessment Research Network member sites reported ENPLs for initial entrustment and at time of graduation. From AYs 2015-2016 to 2017-2018, programs reported ANPLs for initial entrustment decisions. ENPLs and ANPLs were compared using a logistic mixed effects model. RESULTS: ENPLs for graduation and entrustment were most often level 3 (competent) followed by level 4 (proficient). For 8 EPAs, the ENPLs for graduation and entrustment were the same. For the remaining 9, some programs would entrust residents before graduation or graduate them before entrusting them. There were 4,266 supervision level reports for initial entrustment for which an ANPL was provided. ANPLs that were lower than the ENPLs were significantly more likely to be assigned to the medical home-well child (OR = 0.39; 95% CI: 0.26-0.57), transition to adult care (OR = 0.43; 95% CI: 0.19-0.95), behavioral or mental health (OR = 0.36; 95% CI: 0.18-0.71), make referrals (OR = 0.31; 95% CI: 0.17-0.55), lead a team (OR = 0.34; 95% CI: 0.22-0.52), and handovers (OR = 0.18; 95% CI: 0.09-0.36) EPAs. CONCLUSIONS: CCCs reported lower ENPLs for graduation than for entrustment for 5 EPAs, possibly indicating curricular gaps that milestones and EPAs could help identify.

Validation of a QuEChERS-based gas chromatographic method for analysis of pesticide residues in Cassia angustifolia (senna).

A simple multi-residue method based on modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach was established for the determination of 17 organochlorine (OC), 15 organophosphorous (OP) and 7 synthetic pyrethroid (SP) pesticides in an economically important medicinal plant of India, Senna (Cassia angustifolia), by gas chromatography coupled to electron capture and flame thermionic detectors (GC/ECD/FTD) and confirmation of residues was done on gas chromatograph coupled with mass spectrometry (GC-MS). The developed method was validated by testing the following parameters: linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effect, accuracy-precision and measurement uncertainty; the validation study clearly demonstrated the suitability of the method for its intended application. All pesticides showed good linearity in the range 0.01-1.0 µg mL(-1) for OCs and OPs and 0.05-2.5 µg mL(-1) for SPs with correlation coefficients higher than 0.98. The method gave good recoveries for most of the pesticides (70-120%) with intra-day and inter-day precision < 20% in most of the cases. The limits of detection varied from 0.003 to 0.03 mg kg(-1), and the LOQs were determined as 0.01-0.049 mg kg(-1). The expanded uncertainties were <30%, which was distinctively less than a maximum default value of ±50%. The proposed method was successfully applied to determine pesticide residues in 12 commercial market samples obtained from different locations in India.

Low Dose Iron Treatments Induce a DNA Damage Response in Human Endothelial Cells within Minutes.

BACKGROUND: Spontaneous reports from patients able to report vascular sequelae in real time, and recognition that serum non transferrin bound iron may reach or exceed 10µmol/L in the blood stream after iron tablets or infusions, led us to hypothesize that conventional iron treatments may provoke acute vascular injury. This prompted us to examine whether a phenotype could be observed in normal human endothelial cells treated with low dose iron. METHODOLOGY: Confluent primary human endothelial cells (EC) were treated with filter-sterilized iron (II) citrate or fresh media for RNA sequencing and validation studies. RNA transcript profiles were evaluated using directional RNA sequencing with no pre-specification of target sequences. Alignments were counted for exons and junctions of the gene strand only, blinded to treatment types. PRINCIPAL FINDINGS: Rapid changes in RNA transcript profiles were observed in endothelial cells treated with 10µmol/L iron (II) citrate, compared to media-treated cells. Clustering for Gene Ontology (GO) performed on all differentially expressed genes revealed significant differences in biological process terms between iron and media-treated EC, whereas 10 sets of an equivalent number of randomly selected genes from the respective EC gene datasets showed no significant differences in any GO terms. After 1 hour, differentially expressed genes clustered to vesicle mediated transport, protein catabolism, and cell cycle (Benjamini p = 0.0016, 0.0024 and 0.0032 respectively), and by 6 hours, to cellular response to DNA damage stimulus most significantly through DNA repair genes FANCG, BLM, and H2AFX. Comet assays demonstrated that 10µM iron treatment elicited DNA damage within 1 hour. This was accompanied by a brisk DNA damage response pulse, as ascertained by the development of DNA damage response (DDR) foci, and p53 stabilization. SIGNIFICANCE: These data suggest that low dose iron treatments are sufficient to modify the vascular endothelium, and induce a DNA damage response.

Pseudotetraploid clone with structural chromosomal rearrangements in a chondromyxoid fibroma: a case report.

Chondromyxoid fibroma is a rare benign tumor accounting for 1-2% of primary bone tumors. Most of the patients are young males in the 2nd and 3rd decades of life. Metaphyses of long bones are predominantly affected. The histology of this tumor is well established, but its genetic mechanism remains poorly characterized. To our knowledge, only 22 abnormal cytogenetic analyses have been reported, and all contained diploidy or near-diploidy karyograms as their primary event, and inv(6)(p25)(q13) and rearrangements involving regions 6p23-25, 6q12-15, and 6q23-27 constituted a recurrent observation. In this report, a pseudotetraploidy tumor clone with multiple numerical and structural aberrations involving 6p23 as well as other chromosomal loci was identified in a chondromyxoid fibroma from the metaphysis of the left fibula of an 18-year-old male, which has not been reported. The finding may relate to the atypical-looking large cells often seen in this benign tumor.

Clonal trisomy 4 cells detected in the ossifying renal tumor of infancy: study of 3 cases.

The ossifying renal tumor of infancy is a rare neoplasm diagnosed in the first 2 years of life, predominantly in boys. The neoplasm is primarily characterized by the presence of a large ossifying component. Its most common mode of presentation is hematuria, and it has a uniformly benign behavior. The karyotypic makeup of the process has not been reported. Thus, a study was undertaken and it allowed demonstration of clonal trisomy 4, which was confirmed by the fluorescent in-situ hybridization-probing of two additional archival formalin-fixed, paraffin-imbedded similar tumors. On the basis of the findings in these three cases, it seems that clonal trisomy 4 may be considered as a characteristic of the tumor, which makes it distinct from any other infantile renal tumor.

Glycosaminoglycan-mediated selective changes in the aggregation states, zeta potentials, and intrinsic stability of liposomes.

Though the aggregation of glycosaminoglycans (GAGs) in the presence of liposomes and divalent cations has been previously reported, the effects of different GAG species and minor changes in GAG composition on the aggregates that are formed are yet unknown. If minor changes in GAG composition produce observable changes in the liposome aggregate diameter or zeta potential, such a phenomenon may be used to detect potentially dangerous oversulfated contaminants in heparin. We studied the mechanism of the interactions between heparin and its oversulfated glycosaminoglycan contaminants with liposomes. Herein, we demonstrate that Mg(2+) acts to shield the incoming glycosaminoglycans from the negatively charged phosphate groups of the phospholipids and that changes in the aggregate diameter and zeta potential are a function of the glycosaminoglycan species and concentration as well as the liposome bilayer composition. These observations are supported by TEM studies. We have shown that the organizational states of the liposome bilayers are influenced by the presence of GAG and excess Mg(2+), resulting in a stabilizing effect that increases the T(m) value of DSPC liposomes; the magnitude of this effect is also dependent on the GAG species and concentration present. There is an inverse relationship between the percent change in aggregate diameter and the percent change in aggregate zeta potential as a function of GAG concentration in solution. Finally, we demonstrate that the diameter and zeta potential changes in POPC liposome aggregates in the presence of different oversulfated heparin contaminants at low concentrations allow for an accurate detection of oversulfated chondroitin sulfate at concentrations of as low as 1 mol %.

FOXO1-FGFR1 fusion and amplification in a solid variant of alveolar rhabdomyosarcoma.

Rhabdomyosarcoma is the most common pediatric soft tissue malignancy. Two major subtypes, alveolar rhabdomyosarcoma and embryonal rhabdomyosarcoma, constitute 20 and 60% of all cases, respectively. Approximately 80% of alveolar rhabdomyosarcoma carry two signature chromosomal translocations, t(2;13)(q35;q14) resulting in PAX3-FOXO1 fusion, and t(1;13)(p36;q14) resulting in PAX7-FOXO1 fusion. Whether the remaining cases are truly negative for gene fusion has been questioned. We are reporting the case of a 9-month-old girl with a metastatic neck mass diagnosed histologically as solid variant alveolar rhabdomyosarcoma. Chromosome analysis showed a t(8;13;9)(p11.2;q14;9q32) three-way translocation as the sole clonal aberration. Fluorescent in situ hybridization (FISH) demonstrated a rearrangement at the FOXO1 locus and an amplification of its centromeric region. Single-nucleotide polymorphism-based microarray analysis illustrated a co-amplification of the FOXO1 gene at 13q14 and the FGFR1 gene at 8p12p11.2, suggesting formation and amplification of a chimerical FOXO1-FGFR1 gene. This is the first report to identify a novel fusion partner FGFR1 for the known anchor gene FOXO1 in alveolar rhabdomyosarcoma.

Increased secretion of lipoproteins in transgenic mice expressing human D374Y PCSK9 under physiological genetic control.

OBJECTIVE: To produce transgenic mice expressing the D374Y variant of the human proprotein convertase subtilisin/kexin type 9 (PCSK9) gene at physiological levels to investigate the mechanisms causing hypercholesterolemia and accelerated atherosclerosis. METHODS AND RESULTS: A bacterial artificial chromosome containing PCSK9 and its flanking regions was modified to introduce the D374Y mutation and a C-terminal myc(2) tag. Transgenic mice that expressed 1 copy of the mutant or wild-type (WT) PCSK9 bacterial artificial chromosome were produced. Human PCSK9 mRNA was expressed at levels comparable to endogenous pcsk9 and with the same tissue specificity. The expression of D374Y or WT human PCSK9 increased the serum cholesterol level and reduced hepatic low-density lipoprotein receptor protein levels in the transgenic mice compared with bacterial artificial chromosome-negative controls; however, the effects were more marked in D374Y mice. The effect of a high-cholesterol diet on increasing serum cholesterol level was greater in D374Y mice, and atherosclerotic plaques after 15 weeks were more extensive in mice expressing D374Y than in WT PCSK9. D374Y mice secreted more triglyceride-rich lipoproteins into the circulation than WT mice. CONCLUSIONS: The expression of human D374Y PCSK9 at physiological levels produced a phenotype that closely matched that found in heterozygous D374Y patients and suggested that reduced low-density lipoprotein receptor activity is not the sole cause of their hypercholesterolemia.

Degradation of LDLR protein mediated by 'gain of function' PCSK9 mutants in normal and ARH cells.

Dominant gain-of-function mutations in proprotein convertase subtilisin kexin type 9 (PCSK9) cause familial hypercholesterolaemia (FH) and result in accelerated atherosclerosis and premature coronary heart disease. It is believed that PCSK9 binds to LDL-receptor (LDLR) protein and prevents its recycling to the cell surface; gain-of-function PCSK9 mutants enhance LDLR degradation. Several new variants of PCSK9 have been identified, but their effect on PCSK9 activity has not been determined. We describe a new procedure for assessing the activity of four putative gain-of-function mutations identified in FH patients (D129N, D374H, N425S, R496W). All four mutant proteins were secreted normally from transfected HEK293T cells. Immortalized lymphocytes from normolipaemic controls were incubated with conditioned medium from transfected cells and cell-surface LDLR protein was determined by FACS. D374H was as potent as D374Y in reducing cell-surface LDLR, while the other three mutations were more potent than wild type, but less so than the D374 mutants; this correlated with total serum cholesterol in the patients. Substitution of different amino acids at 374 showed that aspartate in this position was critical; even glutamate at residue 374 increased LDLR degradation. When the assay was carried out with ARH-negative lymphocytes that are unable to internalise the LDLR, D374Y-PCSK9 was able to reduce cell-surface LDLR by 35%, compared with approximately 70% for normal lymphocytes. Thus, PCSK9-mediated LDLR degradation is not entirely dependent on ARH function. We propose a novel ARH-independent pathway for PCSK9 activity on LDLR.

Adaptor protein disabled-2 modulates low density lipoprotein receptor synthesis in fibroblasts from patients with autosomal recessive hypercholesterolaemia.

Autosomal recessive hypercholesterolaemia (ARH), characterized clinically by severe inherited hypercholesterolaemia, is caused by recessive null mutations in LDLRAP1 (formerly ARH). Immortalized lymphocytes and monocyte-macrophages, and presumably hepatocytes, from ARH patients fail to take up and degrade plasma low density lipoproteins (LDL) because they lack LDLRAP1, a cargo-specific adaptor required for clathrin-mediated endocytosis of the LDL receptor. Surprisingly, LDL-receptor function is normal in ARH patients' skin fibroblasts in culture. Disabled-2 (Dab2) has been implicated previously in clathrin-mediated internalization of LDL-receptor family members, and we show here that Dab2 is highly expressed in skin fibroblasts, but not in lymphocytes. SiRNA-depletion of Dab2 profoundly reduced LDL-receptor activity in ARH fibroblasts as a result of profound reduction in LDL-receptor protein, but not mRNA; heterologous expression of murine Dab2 reversed this effect. In contrast, LDL-receptor protein content was unchanged in Dab-2-depleted control cells. Incorporation of 35S-labelled amino acids into LDL receptor protein revealed a corresponding apparent reduction in accumulation of newly synthesized LDL-receptor protein on depletion of Dab2 in ARH, but not in control, cells. This reduction in LDL-receptor protein in Dab2-depleted ARH cells could not be reversed by treatment of the cells with proteasomal or lysosomal inhibitors. Thus, we propose a novel role for Dab2 in ARH fibroblasts, where it is apparently required to allow normal translation of LDL receptor mRNA.

A new pepsin assay to detect pulmonary aspiration of gastric contents among newly intubated patients.

Aspiration of gastric contents by endotracheally intubated patients is associated with significant morbidity and mortality. Previous studies suggest that pepsin in tracheal aspirates may be a valuable marker of occult aspiration. We sought to show the sensitivity and specificity of a new, pepsin-specific assay in humans. A prospective, case-controlled study was conducted with subjects serving as their own controls. After planned endotracheal and nasogastric intubation for elective surgery, 20 participants had tracheal and gastric aspirates withdrawn. A blinded investigator tested samples for the presence of pepsin using the assay. Positive samples were then tested with pepstatin, a specific pepsin inhibitor, to ensure that positive results were due to pepsin. All tracheal aspirates tested negative and all gastric aspirates tested positive for pepsin. Pepstatin halted pepsin activity in all positive samples, ensuring that positive results were due to pepsin. A pepsin-specific assay is extremely reliable for detecting gastric contents in humans.

Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1.

Familial hypercholesterolemia is an autosomal dominant disorder with a gene-dosage effect that is usually caused by mutations in the LDL receptor gene that disrupt normal clearance of LDL. In the homozygous form, it results in a distinctive clinical phenotype, characterized by inherited hypercholesterolemia, cholesterol deposition in tendons, and severe premature coronary disease. We described previously two families with autosomal recessive hypercholesterolemia that is not due to mutations in the LDL receptor gene but is characterized by defective LDL receptor-dependent internalization and degradation of LDL by transformed lymphocytes from the patients. We mapped the defective gene to chromosome 1p36 and now show that the disorder in these and a third English family is due to novel mutations in ARH1, a newly identified gene encoding an adaptor-like protein. Cultured skin fibroblasts from affected individuals exhibit normal LDL receptor activity, but their monocyte-derived macrophages are similar to transformed lymphocytes, being unable to internalize and degrade LDL. Retroviral expression of normal human ARH1 restores LDL receptor internalization in transformed lymphocytes from an affected individual, as demonstrated by uptake and degradation of (125)I-labeled LDL and confocal microscopy of cells labeled with anti-LDL-receptor Ab.