Reanalysis of RNA sequencing data ends diagnostic odyssey and expands the phenotypic spectrum of congenital titinopathy.

Although next-generation sequencing has enabled diagnoses for many patients with Mendelian disorders, the majority remain undiagnosed. Here, we present a sibling pair who were clinically diagnosed with Escobar syndrome, however targeted gene testing was negative. Exome sequencing (ES), and later genome sequencing (GS), revealed compound heterozygous TTN variants in both siblings, a maternally inherited frameshift variant [(NM_133378.4):c.36812del; p.(Asp12271Valfs*10)], and a paternally inherited missense variant [(NM_133378.4):c.12322G > A; p.(Asp4108Asn)]. This result was considered nondiagnostic due to poor clinical fit and limited pathogenicity evidence for the missense variant of uncertain significance (VUS). Following initial nondiagnostic RNA sequencing (RNAseq) on muscle and further pursuit of other variants detected on the ES/GS, a reanalysis of noncanonical splice sites in the muscle transcriptome identified an out-of-frame exon retraction in TTN, near the known VUS. Interim literature included reports of patients with similar TTN variants who had phenotypic concordance with the siblings, and a diagnosis of a congenital titinopathy was given 4 years after the TTN variants had been initially reported. This report highlights the value of reanalysis of RNAseq with a different approach, expands the phenotypic spectrum of congenital titinopathy and also illustrates how a perceived phenotypic mismatch, and failure to consider known variants, can result in a prolongation of the diagnostic journey.

Urethral Mesh Assessment in Cancer Patients.

Urethral mesh placement has become a common surgical intervention for the management of stress urinary incontinence. While this procedure offers significant benefits, it is not without potential complications. This review article aims to provide a comprehensive overview of urethral mesh assessment in oncologic patients. The article explores normal magnetic resonance imaging (MRI) and computed tomography (CT) mesh appearances and highlights the pathological aspects associated with urethral mesh complications including both short-term and long-term post-operative complications. By understanding the spectrum of normal findings of urethral mesh and the possible complications, clinicians can improve patient outcomes and make informed decisions regarding urethral mesh management in this patient population.

Outside the fiber: Endomysial stromal and capillary pathology in skeletal muscle may impede infusion therapy in infantile-onset Pompe disease.

The survival of infantile-onset Pompe disease (IOPD) patients has improved dramatically since the introduction of enzyme replacement therapy (ERT) with a1glucosidase alfa. However, long-term IOPD survivors on ERT demonstrate motor deficits indicating that current therapy cannot completely prevent disease progression in skeletal muscle. We hypothesized that in IOPD, skeletal muscle endomysial stroma and capillaries would show consistent changes that could impede the movement of infused ERT from blood to muscle fibers. We retrospectively examined 9 skeletal muscle biopsies from 6 treated IOPD patients using light and electron microscopy. We found consistent ultrastructural endomysial stromal and capillary changes. The endomysial interstitium was expanded by lysosomal material, glycosomes/glycogen, cellular debris, and organelles, some exocytosed by viable muscle fibers and some released on fiber lysis. Endomysial scavenger cells phagocytosed this material. Mature fibrillary collagen was seen in the endomysium, and both muscle fibers and endomysial capillaries showed basal laminar reduplication and/or expansion. Capillary endothelial cells showed hypertrophy and degeneration, with narrowing of the vascular lumen. Ultrastructurally defined stromal and vascular changes likely constitute obstacles to movement of infused ERT from capillary lumen to muscle fiber sarcolemma, contributing to the incomplete efficacy of infused ERT in skeletal muscle. Our observations can inform approaches to overcoming these barriers to therapy.

Cytostatic hypothermia and its impact on glioblastoma and survival.

Patients with glioblastoma (GBM) have limited options and require novel approaches to treatment. Here, we studied and deployed nonfreezing "cytostatic" hypothermia to stunt GBM growth. This growth-halting method contrasts with ablative, cryogenic hypothermia that kills both neoplastic and infiltrated healthy tissue. We investigated degrees of hypothermia in vitro and identified a cytostatic window of 20° to 25°C. For some lines, 18 hours/day of cytostatic hypothermia was sufficient to halt division in vitro. Next, we fabricated an experimental tool to test local cytostatic hypothermia in two rodent GBM models. Hypothermia more than doubled median survival, and all rats that successfully received cytostatic hypothermia survived their study period. Unlike targeted therapeutics that are successful in preclinical models but fail in clinical trials, cytostatic hypothermia leverages fundamental physics that influences biology broadly. It is a previously unexplored approach that could provide an additional option to patients with GBM by halting tumor growth.

Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1-3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Successful Neonatal, Intraoperative Neuromonitoring in the Surgical Correction of a Thoracic Dermal Sinus Tract: Technical Note.

INTRODUCTION: Intraoperative neuromonitoring (IONM) is commonly used during surgery of the spine and spinal cord for early surveillance of iatrogenic injury to the central and peripheral nervous system. However, for infants and young children under 3 years of age, the use of IONM is challenging due to incomplete central and peripheral myelination. CASE PRESENTATION: We report a case of a T4-T6 dermal sinus tract (DST) that was resected on day of life 23, with the successful use of IONM. CONCLUSION: To our knowledge, this is the youngest reported case of the use of IONM in the surgical correction of a DST in a neonatal patient. This case demonstrates the potential efficacy of IONM in neonatal spine surgery and the techniques used to adapt the technology to an immature nervous system.

Somatic variants in diverse genes leads to a spectrum of focal cortical malformations.

Post-zygotically acquired genetic variants, or somatic variants, that arise during cortical development have emerged as important causes of focal epilepsies, particularly those due to malformations of cortical development. Pathogenic somatic variants have been identified in many genes within the PI3K-AKT-mTOR-signalling pathway in individuals with hemimegalencephaly and focal cortical dysplasia (type II), and more recently in SLC35A2 in individuals with focal cortical dysplasia (type I) or non-dysplastic epileptic cortex. Given the expanding role of somatic variants across different brain malformations, we sought to delineate the landscape of somatic variants in a large cohort of patients who underwent epilepsy surgery with hemimegalencephaly or focal cortical dysplasia. We evaluated samples from 123 children with hemimegalencephaly (n = 16), focal cortical dysplasia type I and related phenotypes (n = 48), focal cortical dysplasia type II (n = 44), or focal cortical dysplasia type III (n = 15). We performed high-depth exome sequencing in brain tissue-derived DNA from each case and identified somatic single nucleotide, indel and large copy number variants. In 75% of individuals with hemimegalencephaly and 29% with focal cortical dysplasia type II, we identified pathogenic variants in PI3K-AKT-mTOR pathway genes. Four of 48 cases with focal cortical dysplasia type I (8%) had a likely pathogenic variant in SLC35A2. While no other gene had multiple disease-causing somatic variants across the focal cortical dysplasia type I cohort, four individuals in this group had a single pathogenic or likely pathogenic somatic variant in CASK, KRAS, NF1 and NIPBL, genes previously associated with neurodevelopmental disorders. No rare pathogenic or likely pathogenic somatic variants in any neurological disease genes like those identified in the focal cortical dysplasia type I cohort were found in 63 neurologically normal controls (P = 0.017), suggesting a role for these novel variants. We also identified a somatic loss-of-function variant in the known epilepsy gene, PCDH19, present in a small number of alleles in the dysplastic tissue from a female patient with focal cortical dysplasia IIIa with hippocampal sclerosis. In contrast to focal cortical dysplasia type II, neither focal cortical dysplasia type I nor III had somatic variants in genes that converge on a unifying biological pathway, suggesting greater genetic heterogeneity compared to type II. Importantly, we demonstrate that focal cortical dysplasia types I, II and III are associated with somatic gene variants across a broad range of genes, many associated with epilepsy in clinical syndromes caused by germline variants, as well as including some not previously associated with radiographically evident cortical brain malformations.

Resolution of radiation necrosis with bevacizumab following radiation therapy for primary CNS lymphoma.

IMPORTANCE: Radiation necrosis (RN) is a rare but serious adverse effect following treatment with radiation therapy. No standard of care exists for the management of RN, and efforts to prevent and treat RN are limited by a lack of insight into the pathomechanics and molecular drivers of RN. This case series describes the outcomes of treatment with bevacizumab (BV) in two primary CNS lymphoma (PCNSL) patients who developed symptomatic biopsy-proven RN after whole brain radiation (WBRT) with a stereotactic radiosurgery (SRS) boost. OBSERVATIONS: Patient 1 is a 52 year-old female with PCNSL treated with WBRT followed by an SRS boost. She developed symptomatic biopsy-proven RN, and initial treatment with tocopherol and pentoxifylline was unsuccessful. A 100% clinical and radiographic response was achieved with 4 cycles of BV. Patient 2, a 48 year-old male with PCNSL, presented with seizures and biopsy-proven RN after radiation therapy. Initial empiric treatment with tocopherol and pentoxifylline was unsuccessful. A 100% clinical and radiographic response was achieved with 3 cycles of BV. CONCLUSIONS AND RELEVANCE: Monitoring for RN in patients with PCNSL treated with radiation therapy is warranted. BV is an efficacious treatment and a viable alternative to corticosteroids or surgical intervention.

Wastewater sequencing uncovers early, cryptic SARS-CoV-2 variant transmission.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing/sequencing capacity, which can also introduce biases. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here, we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We develop and deploy improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detect emerging variants of concern up to 14 days earlier in wastewater samples, and identify multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Case Report: Unusual Aggregation of Different Glomerulopathies in a Family Resolved by Genetic Testing and Reverse Phenotyping.

Glomerular diseases (GDs) are a major cause of chronic kidney disease in children. The conventional approach to diagnosis of GDs includes clinical evaluation and, in most cases, kidney biopsy to make a definitive diagnosis. However, in many cases, clinical presentations of different GDs can overlap, leading to uncertainty in diagnosis and management even after renal biopsy. In this report, we identify a family with clinical diagnoses of postinfectious glomerulonephritis and IgA nephropathy in a parent and two children. Renal biopsies were initially inconclusive; however, genetic testing showed that the two individuals diagnosed at different points with IgA nephropathy carried novel segregating pathogenic variants in COL4A5 gene. We were only able to make the final diagnoses in each of the family members after genetic testing and reverse phenotyping. This case highlights the utility of genetic testing and reverse phenotyping in resolving clinical diagnosis in families with unusual constellations of different glomerulopathies. We propose that clustering of different glomerular disease phenotypes in a family should be an indication for genetic testing followed by reverse phenotyping.

Blockade of the natriuretic peptide clearance receptor attenuates proteinuria in a mouse model of focal segmental glomerulosclerosis.

Glomerular podocytes play a key role in proteinuric diseases. Accumulating evidence suggests that cGMP signaling has podocyte protective effects. The major source of cGMP generation in podocytes is natriuretic peptides. The natriuretic peptide clearance receptor (NPRC) binds and degrades natriuretic peptides. As a result, NPRC inhibits natriuretic peptide-induced cGMP generation. To enhance cGMP generation in podocytes, we blocked natriuretic peptide clearance using the specific NPRC ligand ANP(4-23). We then studied the effects of NPRC blockade in both cultured podocytes and in a mouse transgenic (TG) model of focal segmental glomerulosclerosis (FSGS) created in our laboratory. In this model, a single dose of the podocyte toxin puromycin aminonucleoside (PAN) causes robust albuminuria in TG mice, but only mild disease in non-TG animals. We found that natriuretic peptides protected cultured podocytes from PAN-induced apoptosis, and that ANP(4-23) enhanced natriuretic peptide-induced cGMP generation in vivo. PAN-induced heavy proteinuria in vehicle-treated TG mice, and this increase in albuminuria was reduced by treatment with ANP(4-23). Treatment with ANP(4-23) also reduced the number of mice with glomerular injury and enhanced urinary cGMP excretion, but these differences were not statistically significant. Systolic BP was similar in vehicle and ANP(4-23)-treated mice. These data suggest that: 1. Pharmacologic blockade of NPRC may be useful for treating glomerular diseases such as FSGS, and 2. Treatment outcomes might be improved by optimizing NPRC blockade to inhibit natriuretic peptide clearance more effectively.

Hemodynamic and structural brain measures in high and low sedentary older adults.

Due to its cardiovascular effects sedentary behaviour might impact cerebrovascular function in the long term, affecting cerebrovascular regulatory mechanisms and perfusion levels. Consequently this could underly potential structural brain abnormalities associated with cognitive decline. We therefore assessed the association between sedentary behaviour and brain measures of cerebrovascular perfusion and structural abnormalities in community-dwelling older adults. Using accelerometery (GENEActiv) data from The Irish Longitudinal Study on Ageing (TILDA) we categorised individuals by low- and high-sedentary behaviour (≤8 vs >8 hours/day). We examined prefrontal haemoglobin oxygenation levels using Near-Infrared Spectroscopy during rest and after an orthostatic challenge in 718 individuals (66 ± 8 years, 52% female). Global grey matter cerebral blood flow, total grey and white matter volume, total and subfield hippocampal volumes, cortical thickness, and white matter hyperintensities were measured using arterial spin labelling, T1, and FLAIR MRI in 86 individuals (72 ± 6 years, 55% female). While no differences in prefrontal or global cerebral hemodynamics were found between groups, high-sedentary individuals showed lower hippocampal volumes and increased white matter hyperintensities compared to their low-sedentary counterparts. Since these structural cerebral abnormalities are associated with cognitive decline and Alzheimer's disease, future work exploring the causal pathways underlying these differences is needed.

Multisite Quality Improvement Study of a Patient-Pathologist Consultation Program.

OBJECTIVES: The aim of this multisite quality improvement study was to evaluate patients' experiences with the patient-centered pathology (PCP) consultation program and to determine whether PCP enhanced their care experience. METHODS: Patients were invited to attend PCP consultations to review their pathology report and slides and have their questions answered by the pathologist privately, with the option to attend the appointment with family members or friends for support. A patient experience questionnaire (PEQ) was administered to patients, who participated voluntarily in the PCP, and survey data were collected and stored in REDCap. Statistical analysis was performed using SAS 9.4 (SAS Institute). RESULTS: Sixty-seven patients (95.5% female) aged 18 to 84 years across 4 institutions completed the PEQ. Overall, 58% and 15.8% of patients had breast and brain tumors, respectively, and 59.7% of tumors were newly diagnosed. Most patients thought it was important for them to learn as much as they could about their health condition. However, the majority of patients reported some degree of difficulty learning about their health condition based on written information, despite 97% having completed high school and/or further education. The majority of patients rated their pathologist as "excellent" across communication metrics. Ultimately, 100% of respondents were satisfied, found their visits to be useful, and would recommend the PCP to other patients. CONCLUSIONS: Patients found that personalized clinical encounters with pathologists improved their understanding of their health condition and their satisfaction with their care experience. Patients thought pathologists communicated respectfully, effectively, and empathetically.

Eosinophilic globules in a classic ependymoma: evidence of a possible secretory role.

A number of neoplasms of the central nervous system can demonstrate diffuse eosinophilic globules, known to be secretory products of the corresponding cell type, but they have not been a salient feature in descriptions of classic ependymoma. Here, we present a case of a posterior fossa ependymoma demonstrating glassy PAS-positive, diastase-resistant, eosinophilic globules with light microscopic and ultrastructural features resembling Reissner fiber, the secretory product of the subcommissural organ. While there has been a single published description of an ependymoma with intra- and extracellular granulofibrillary material suggested to be evidence of secretory differentiation, ours is the first case to demonstrate diffuse eosinophilic globules in an ependymoma. The extent of globules allowed full study by electron microscopy to provide new insight into the secretory material and the surrounding structures. Our findings suggest that neoplastic ependymal cells can recapitulate the secretory capacity of the subcommissural organ.

Is there an association between orthostatic hypotension and cerebral white matter hyperintensities in older people? The Irish longitudinal study on ageing.

INTRODUCTION: Orthostatic Hypotension (OH) is an abnormal drop in blood pressure (BP) that occurs following orthostatic challenge. OH is associated with increased risk of falls, cognitive impairment and death. White Matter Hyperintensities (WMH) on MR Brain are associated with vascular risk factors such as hypertension, diabetes and age. We examined whether extent White matter intensities were associated with presence of OH detected in a community dwelling population of older people. METHODS: Individuals from the MR sub-study of the Irish Longitudinal Study of Ageing underwent a 3 Tesla MR Brain scan to assess WMH severity (Schelten's Score). The scans were performed during the Wave 3 TILDA health assessment phase when the subjects also underwent assessment for OH with an active stand protocol. Data was analysed for association between WMH and vascular risks and orthostatic change in BP 10 second intervals during the OH evaluation. RESULTS: 440 subjects were investigated; median age 72 years (65-92 years) and 228 (51.5%) female. Range of Scheltens' Scores was 0-32. Mean score was 9.72 (SD 5.87). OH was detected in 68.4% (301). On linear regression, positive associations were found between Scheltens' Score and age, hypertension, prior history of stroke and TIA, and with OH at 30, 70, 90 and 100 seconds following standing (p < 0.05, O.R. 1.9-2.5). CONCLUSION: WMD is associated with OH detected at multiple time points using active stand in community dwelling older subjects. Further research is necessary to evaluate the direction of this association.

Adenocarcinoma of the lung: from BAC to the future.

Adenocarcinoma in situ, minimally invasive adenocarcinoma, lepidic predominant adenocarcinoma and invasive mucinous adenocarcinoma are relatively new classification entities which replace the now retired term, bronchoalveolar carcinoma (BAC). The radiographic appearance of these lesions ranges from pure, ground glass nodules to large, solid masses. A thorough understanding of the new classification is essential to radiologists who work with MDT colleagues to provide accurate staging and treatment. A 2-year review was performed of all surgically resected cases of adenocarcinoma in situ, minimally invasive adenocarcinoma and lepidic predominant adenocarcinoma in our institution. Cases are broken down by age, gender, tumour type and tumour location. A pictorial review is presented to illustrate the radiologic and pathologic features of each entity.