Compound heterozygous splicing variants expand the genotypic spectrum of EMC1-related disorders.

EMC1 encodes subunit 1 of the endoplasmic reticulum (ER) membrane protein complex (EMC), a transmembrane domain insertase involved in membrane protein biosynthesis. Variants in EMC1 are described as a cause of global developmental delay, hypotonia, cortical visual impairment, and commonly, cerebral atrophy on MRI scan. We report an individual with severe global developmental delay and progressive cerebellar atrophy in whom exome sequencing identified a heterozygous essential splice-site variant in intron-3 of EMC1 (NM_015047.3:c.287-1G>A). Whole genome sequencing (WGS) identified a deep intronic variant in intron-20 of EMC1 (NM_015047.3:c.2588-771C>G) that was poorly predicted by in silico programs to disrupt pre-mRNA splicing. Reverse Transcription-PCR (RT-PCR) revealed stochastic activation of a pseudo-exon associated with the c.2588-771C>G variant and mis-splicing arising from the c.287-1G>A variant. This case highlights the utility of WGS and RNA studies to identify and assess likely pathogenicity of deep intronic variants and expands the genotypic and phenotypic spectrum of EMC1-related disorders.

The Role of Cue-Based Strategies in Skilled Diagnosis Among Pathologists.

OBJECTIVE: This research was designed to test whether behavioral indicators of pathology-related cue utilization were associated with performance on a diagnostic task. BACKGROUND: Across many domains, including pathology, successful diagnosis depends on pattern recognition that is supported by associations in memory in the form of cues. Previous studies have focused on the specific information or knowledge on which medical image expertise relies. The target in this study is the more general ability to identify and interpret relevant information. METHOD: Data were collected from 54 histopathologists in both conference and online settings. The participants completed a pathology edition of the Expert Intensive Skills Evaluation 2.0 (EXPERTise 2.0) to establish behavioral indicators of context-related cue utilization. They also completed a separate diagnostic task designed to examine related diagnostic skills. RESULTS: Behavioral indicators of higher or lower cue utilization were based on the participants' performance across five tasks. Accounting for the number of cases reported per year, higher cue utilization was associated with greater accuracy on the diagnostic task. A post hoc analysis suggested that higher cue utilization may be associated with a greater capacity to recognize low prevalence cases. CONCLUSION: This study provides support for the role of cue utilization in the development and maintenance of skilled diagnosis amongst pathologists. APPLICATION: Pathologist training needs to be structured to ensure that learners have the opportunity to form cue-based strategies and associations in memory, especially for less commonly seen diseases.

Hereditary diffuse gastric cancer: updated clinical practice guidelines.

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome that is characterised by a high prevalence of diffuse gastric cancer and lobular breast cancer. It is largely caused by inactivating germline mutations in the tumour suppressor gene CDH1, although pathogenic variants in CTNNA1 occur in a minority of families with HDGC. In this Policy Review, we present updated clinical practice guidelines for HDGC from the International Gastric Cancer Linkage Consortium (IGCLC), which recognise the emerging evidence of variability in gastric cancer risk between families with HDGC, the growing capability of endoscopic and histological surveillance in HDGC, and increased experience of managing long-term sequelae of total gastrectomy in young patients. To redress the balance between the accessibility, cost, and acceptance of genetic testing and the increased identification of pathogenic variant carriers, the HDGC genetic testing criteria have been relaxed, mainly through less restrictive age limits. Prophylactic total gastrectomy remains the recommended option for gastric cancer risk management in pathogenic CDH1 variant carriers. However, there is increasing confidence from the IGCLC that endoscopic surveillance in expert centres can be safely offered to patients who wish to postpone surgery, or to those whose risk of developing gastric cancer is not well defined.

Congenital Titinopathy: Comprehensive characterization and pathogenic insights.

OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.

Nemaline myopathy and distal arthrogryposis associated with an autosomal recessive TNNT3 splice variant.

A male neonate presented with severe weakness, hypotonia, contractures and congenital scoliosis. Skeletal muscle specimens showed marked atrophy and degeneration of fast fibers with striking nemaline rods and hypertrophy of slow fibers that were ultrastructurally normal. A neuromuscular gene panel identified a homozygous essential splice variant in TNNT3 (chr11:1956150G > A, NM_006757.3:c.681+1G > A). TNNT3 encodes skeletal troponin-Tfast and is associated with autosomal dominant distal arthrogryposis. TNNT3 has not previously been associated with nemaline myopathy (NM), a rare congenital myopathy linked to defects in proteins associated with thin filament structure and regulation. cDNA studies confirmed pathogenic consequences of the splice variant, eliciting exon-skipping and intron retention events leading to a frameshift. Western blot showed deficiency of troponin-Tfast protein with secondary loss of troponin-Ifast . We establish a homozygous splice variant in TNNT3 as the likely cause of severe congenital NM with distal arthrogryposis, characterized by specific involvement of Type-2 fibers and deficiency of troponin-Tfast .

ß-HCG Elevation in Wilms Tumor: An Uncommon Presentation.

Wilms tumor (nephroblastoma) is a readily diagnosed common abdominal tumor in children. Rarely, it may present with factors that may confound the diagnosis. We report a 6-year-old female child who presented with a rapidly growing and invasive abdominal mass with the histopathologic features of Wilms tumor associated with an elevated serum beta human chorionic gonadotropin, which has not been previously reported in this condition.

Aganglionosis with the absence of hypertrophied nerve fibres predicts disease proximal to rectosigmoid colon.

PURPOSE: The gold standard for the diagnosis of Hirschsprung's disease (HSCR) is the pathologic evaluation of a rectal biopsy that demonstrates the absence of ganglion cells and nerve fibre hypertrophy. However, it has been frequently reported that hypertrophic nerves may not be present in some variants like long-segment HSCR, total colonic aganglionosis, premature and very young infants. The aim of this study was to determine this association. METHODS: We performed a retrospective review of the HSCR database at our tertiary care children's hospital from 2000 to 2013. In order to analyse the relationship between the diameter of the nerve fibres and the level of aganglionosis, we classified the patient sample into two groups-fibres ≤40 and >40 µm. The groups were statistically compared with P < 0.05 being significant. RESULTS: Rectal biopsies of 92 patients confirmed as HSCR with definitive operation performed at the same institution were reviewed. The mean nerve diameter was 50.1 µm (range 20-87.5 µm). Nerve fibre diameter ≤40 µm was predictive of transition zone above the sigmoid colon. A specificity of 77.3 % and a likelihood ratio of 2.03 supported this perception. No correlation was noted between nerve fibre diameter and gestational age at birth, birth weight or age at biopsy. CONCLUSION: The absence of nerve fibre hypertrophy in the presence of aganglionosis on rectal biopsy specimens is predictive of long-segment HSCR.

Testicular microlithiasis: the importance of self-examination.

AIM: To explore the issue of appropriate management of testicular microlithiasis. We report the third ever case of tumour arising from a testis previously known to have microlithiasis in childhood and review the literature to provide an evidence-based approach to management of testicular microlithiasis. METHODS: Case report and review of previous literature. RESULTS AND CONCLUSIONS: Although there is a strong association between testicular microlithiasis and testicular malignancy at diagnosis, there are only three reported cases of subsequent tumour development in childhood. Testicular microlithiasis is an increasingly recognised entity. There is insufficient evidence in the current literature to support any regime of clinical surveillance. Self-examination is the most important factor in the early detection of testicular malignancy.

An optimized 3T MRI scan protocol to assess iris melanoma with subsequent histopathological verification - A prospective study.

INTRODUCTION: Magnetic resonance imaging (MRI) has demonstrated high levels of tissue contrast, accuracy and reproducibility in evaluating posterior uveal melanoma. Owing to smaller size, the role of MRI in detecting and characterising iris melanoma has not yet been explored. AIMS: To develop a protocol to image iris melanoma and describe the MRI characteristics of histopathological-confirmed iris melanoma. MATERIALS AND METHODS: An optimised MRI protocol, using a 3T MRI scanner and a 32-channel head coil, was developed to image iris tumours. A prospective, single-centre, 12-month study was conducted on all patients with lesions suspicious for iris melanoma. All patients were offered an MRI scan in addition to the standardised clinical procedures. Image quality comparison was made with existing clinical investigations. Iris melanoma characteristics on MRI are described. RESULTS: A successful optimised MRI scan protocol was developed that was able to detect and characterise iris melanoma. One normal participant and five patients with subsequent histopathological-confirmed iris melanoma (n = 6) were recruited. Four patients completed the full MRI sequence. All iris melanoma were detected on at least one T1- or T2-weighted images. When compared to the vitreous, all iris melanomas demonstrated hyper-intensity on T1-weighted images and hypo-intensity on T2-weighted images. On T1-mapping, T1-values of iris melanoma demonstrated an inverse relationship with the degree of tumour pigmentation. CONCLUSIONS: This study highlights an optimised, easily reproducible MRI scan protocol to image iris melanoma. Numerous MR imaging characteristics of iris melanoma are reported for the first time and a potential non-invasive tumour biomarker is described.

Faculty development for strengthening online teaching capability: a mixed-methods study of what staff want, evaluated with Kirkpatrick's model of teaching effectiveness.

Background: Globally, tertiary teachers are increasingly being pushed and pulled into online teaching. While most developments in online education have focused on the student perspective, few studies have reported faculty development (FD) initiatives for increasing online teaching capability and confidence from a staff perspective. Methods: We designed and evaluated FD workshops, using five datasets, and the use of H5P software for interactive online teaching. We used educational theory to design our FD (Mayer multimedia principles, active learning) and evaluated our FD initiatives using the Best Evidence Medical Education (BEME) 2006 modified Kirkpatrick levels. Results: Teaching staff reported that Communities of Practice were important for their learning and emotional support. Uptake and deployment of FD skills depended on the interactivity of FD sessions, their timeliness, and sufficient time allocated to attend and implement. Staff who applied FD learning to their online teaching created interactive learning resources. This content was associated with an increase in student grades, and the roll-out of an institutional site-wide H5P license. Conclusion: This paper demonstrates an effective strategy for upskilling and upscaling faculty development. The use of H5P as a teaching tool enhances student learning. For successful FD, we make four recommendations. These are: provide just-in-time learning and allocate time for FD and staff to create online teaching material; foster supportive communities; offer personalized support; and design hands on active learning.

Neonatal lethal Costello syndrome and unusual dinucleotide deletion/insertion mutations in HRAS predicting p.Gly12Val.

De novo heterozygous mutations in HRAS cause Costello syndrome (CS), a condition with high mortality and morbidity in infancy and early childhood due to cardiac, respiratory, and muscular complications. HRAS mutations predicting p.Gly12Val, p.Gly12Asp, and p.Gly12Cys substitutions have been associated with severe, lethal, CS. We report on molecular, clinical, and pathological findings in patients with mutations predicting HRAS p.Gly12Val that were identified in our clinical molecular genetic testing service. Such mutations were identified in four patients. Remarkably, three were deletion/insertion mutations affecting coding nucleotides 35 and 36. All patients died within 6 postnatal weeks, providing further evidence that p.Gly12Val mutations predict a very poor prognosis. High birth weight, polyhydramnios (and premature birth), cardiac hypertrophy, respiratory distress, muscle weakness, and postnatal growth failure were present. Dysmorphism was subtle or non-specific, with edema, coarsened facial features, prominent forehead, depressed nasal bridge, anteverted nares, and low-set ears. Proximal upper limb shortening, a small bell-shaped chest, talipes, and fixed flexion deformities of the wrists were seen. Neonatal atrial arrhythmia, highly suggestive of CS, was also present in two patients. One patient had congenital alveolar dysplasia, and another, born after 36 weeks' gestation, bronchopulmonary dysplasia. A rapidly fatal disease course, and the difficulty of identifying subtle dysmorphism in neonates requiring intensive care, suggest that this condition remains under-recognized, and should enter the differential diagnosis for very sick infants with a range of clinical problems including cardiac hypertrophy and disordered pulmonary development. Clinical management should be informed by knowledge of the poor prognosis of this condition.

Serratia Marcescens Endophthalmitis and Bacteraemia following Complicated Cataract Surgery.

A 93-year-old male presented with left eye pain, fever and loss of vision two days after complicated cataract surgery. A diagnosis of Serratia marcescens endophthalmitis and systemic bacteremia was made after the organism was identified on vitreous and peripheral blood cultures. This case demonstrates that an aggressive intraocular infection can lead to bacteremia.

WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase.

OBJECTIVE: To describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia. METHODS: Exome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle. RESULTS: Splice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%-5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness. CONCLUSIONS: Whole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

Optimizing muscle biopsy for the diagnosis of mitochondrial myopathy.

PURPOSE: To establish a reliable technique for harvesting the orbicularis oculi muscle to facilitate diagnosis of chronic progressive external ophthalmoplegia, a mitochondrial myopathy. METHODS: In this retrospective observational case series, 10 patients clinically suspected to have chronic progressive external ophthalmoplegia underwent surgery for upper eyelid ptosis. A protocol for orbicularis biopsy was developed. Initial cases of levator muscle biopsy yielded inadequate, unorientated skeletal muscle with significant contraction artifact that prevented the study of morphologic features. To improve yield and quality, orbicularis oculi muscle biopsy was performed in the later patients following a standard muscle biopsy protocol used for limb muscles. This involved suturing a third of the muscle on a wooden stick, to keep it at isometric length. The specimen was sent fresh in saline-moistened gauze to the pathologist who then divided the muscle for various studies. RESULTS: The biopsies of orbicularis muscle performed using this protocol resulted in adequate skeletal muscle with an acceptable level of artifact. Mitochondrial myopathy was diagnosed in 9 of 10 cases. CONCLUSIONS: The orbicularis oculi muscle is a good source of skeletal muscle for investigating muscle disorders, and it is easily collected during blepharoplasty or ptosis surgery. This has avoided the need for a standard proximal limb muscle biopsy, thereby reducing morbidity and cost to the patients.

Destabilized adhesion in the gastric proliferative zone and c-Src kinase activation mark the development of early diffuse gastric cancer.

The initial development of diffuse gastric cancer (DGC) is poorly understood. The study of E-cadherin (CDH1) germ line mutation carriers predisposed to DGC provides a rare opportunity to elucidate the genetic and biological events surrounding disease initiation. Samples from various stages of hereditary and sporadic DGC were investigated to determine general mechanisms underlying early DGC development. Paraffin-embedded tissues from 13 CDH1 mutation carriers and from 10 sporadic early DGC cases were analyzed. Immunofluorescence and immunohistochemistry using differentiation, proliferation, and adhesion markers showed that DGC initiation seems to occur at the proliferative zone (the upper neck) of the gastric epithelium and correlates with absent or reduced expression of junctional proteins (beta-actin, p120, Lin-7). Slow proliferation of neoplastic cells at the upper gastric neck leads to the formation of intramucosal signet-ring cell carcinoma (SRCC) displaying differentiated features. As shown by immunolabeling, invasion from SRCC lesions beyond the gastric mucosa is associated with poor differentiation, increased proliferation, activation of the c-Src system, and an epithelial-mesenchymal transition. Our results provide a molecular description of the early development of DGC and explain the relationship between the two main DGC types, poorly differentiated carcinoma and SRCC: both share their origin, but SRCC develops following cancer cell differentiation and seems relatively indolent in its intramucosal stage.

An Unusual Presentation of Pediatric Conjunctival Mucosa-Associated Lymphoid Tissue Lymphoma.

Ocular adnexal mucosa-associated lymphoid tissue (MALT) lymphoma is uncommon in the pediatric population. Initial misdiagnosis is common and there is lacking consensus regarding the optimal approach to treatment. Herein, we report an atypical presentation of pediatric conjunctival MALT lymphoma and review the presentation and management of this rare condition.

Increased SPARC expression in primary angle closure glaucoma iris.

PURPOSE: SPARC (secreted protein, acidic, and rich in cysteine) is involved in extracellular matrix (ECM) organization. The purpose of this study was to evaluate the expression of SPARC in iris tissue from primary angle closure glaucoma (PACG) eyes. METHODS: Iris tissue was obtained from peripheral iridectomies performed during trabeculectomy surgery in nine PACG and 16 primary open-angle glaucoma (POAG) eyes at the Singapore National Eye Centre. Three non-glaucoma control iris specimens were obtained from patients who underwent Descemet's stripping automated endothelial keratoplasty (DSAEK) procedure. SPARC and collagen I expression were quantified by real-time polymerase chain reaction (PCR). The histological distribution of collagen I and III in the iris stroma was determined using picrosirius red polarization. Density of the iris stromal vasculature was also calculated. RESULTS: The mean age was 68.9+/-10.9 years and 65.7+/-12.2 years in POAG and PACG groups, respectively. The PACG iris expressed SPARC 13.6-fold more and collagen I 5.2 fold more compared to non-glaucoma control iris. The PACG iris also demonstrated 3.3 fold higher SPARC and 2.0 fold higher collagen I expression relative to the POAG iris. The density of collagen I was greater in PACG eyes than in POAG and control eyes (p<0.001). The mean density of iris stromal blood vessels per micron square area was similar in all three groups. CONCLUSIONS: SPARC was significantly increased in the PACG iris. The data suggest that SPARC could play a role in the development of PACG by influencing the biomechanical properties of the iris through a change in ECM organization.

Convolutional Deep Belief Network with Feature Encoding for Classification of Neuroblastoma Histological Images.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumor in children younger than 5 years old. Optimal management of neuroblastic tumors depends on many factors including histopathological classification. The gold standard for classification of neuroblastoma histological images is visual microscopic assessment. In this study, we propose and evaluate a deep learning approach to classify high-resolution digital images of neuroblastoma histology into five different classes determined by the Shimada classification. SUBJECTS AND METHODS: We apply a combination of convolutional deep belief network (CDBN) with feature encoding algorithm that automatically classifies digital images of neuroblastoma histology into five different classes. We design a three-layer CDBN to extract high-level features from neuroblastoma histological images and combine with a feature encoding model to extract features that are highly discriminative in the classification task. The extracted features are classified into five different classes using a support vector machine classifier. DATA: We constructed a dataset of 1043 neuroblastoma histological images derived from Aperio scanner from 125 patients representing different classes of neuroblastoma tumors. RESULTS: The weighted average F-measure of 86.01% was obtained from the selected high-level features, outperforming state-of-the-art methods. CONCLUSION: The proposed computer-aided classification system, which uses the combination of deep architecture and feature encoding to learn high-level features, is highly effective in the classification of neuroblastoma histological images.

Computer Aided Classification of Neuroblastoma Histological Images Using Scale Invariant Feature Transform with Feature Encoding.

Neuroblastoma is the most common extracranial solid malignancy in early childhood. Optimal management of neuroblastoma depends on many factors, including histopathological classification. Although histopathology study is considered the gold standard for classification of neuroblastoma histological images, computers can help to extract many more features some of which may not be recognizable by human eyes. This paper, proposes a combination of Scale Invariant Feature Transform with feature encoding algorithm to extract highly discriminative features. Then, distinctive image features are classified by Support Vector Machine classifier into five clinically relevant classes. The advantage of our model is extracting features which are more robust to scale variation compared to the Patched Completed Local Binary Pattern and Completed Local Binary Pattern methods. We gathered a database of 1043 histologic images of neuroblastic tumours classified into five subtypes. Our approach identified features that outperformed the state-of-the-art on both our neuroblastoma dataset and a benchmark breast cancer dataset. Our method shows promise for classification of neuroblastoma histological images.