Identifying cellular signalling molecules in developmental disorders of the brain: Evidence from focal cortical dysplasia and tuberous sclerosis.

AIMS: We understand little of the pathogenesis of developmental cortical lesions, because we understand little of the diversity of the cell types that contribute to the diseases or how those cells interact. We tested the hypothesis that cellular diversity and cell-cell interactions play an important role in these disorders by investigating the signalling molecules in the commonest cortical malformations that lead to childhood epilepsy, focal cortical dysplasia (FCD) and tuberous sclerosis (TS). METHODS: Transcriptional profiling clustered cases into molecularly distinct groups. Using gene expression data, we identified the secretory signalling molecules in FCD/TS and characterised the cell types expressing these molecules. We developed a functional model using organotypic cultures. RESULTS: We identified 113 up-regulated secretory molecules in FCDIIB/TS. The top 12 differentially expressed genes (DEGs) were validated by immunohistochemistry. This highlighted two molecules, Chitinase 3-like protein 1 (CHI3L1) and C-C motif chemokine ligand 2 (CCL2) (MCP1) that were expressed in a unique population of small cells in close proximity to balloon cells (BC). We then characterised these cells and developed a functional model in organotypic slice cultures. We found that the number of CHI3L1 and CCL2 expressing cells decreased following inhibition of mTOR, the main aberrant signalling pathway in TS and FCD. CONCLUSIONS: Our findings highlight previously uncharacterised small cell populations in FCD and TS which express specific signalling molecules. These findings indicate a new level of diversity and cellular interactions in cortical malformations and provide a generalisable approach to understanding cell-cell interactions and cellular heterogeneity in developmental neuropathology.

DNA methylation-based profiling for paediatric CNS tumour diagnosis and treatment: a population-based study.

BACKGROUND: Marked variation exists in the use of genomic data in tumour diagnosis, and optimal integration with conventional diagnostic technology remains uncertain despite several studies reporting improved diagnostic accuracy, selection for targeted treatments, and stratification for trials. Our aim was to assess the added value of molecular profiling in routine clinical practice and the impact on conventional and experimental treatments. METHODS: This population-based study assessed the diagnostic and clinical use of DNA methylation-based profiling in childhood CNS tumours using two large national cohorts in the UK. In the diagnostic cohort-which included routinely diagnosed CNS tumours between Sept 1, 2016, and Sept 1, 2018-we assessed how the methylation profile altered or refined diagnosis in routine clinical practice and estimated how this would affect standard patient management. For the archival cohort of diagnostically difficult cases, we established how many cases could be solved using modern standard pathology, how many could only be solved using the methylation profile, and how many remained unsolvable. FINDINGS: Of 484 patients younger than 20 years with CNS tumours, 306 had DNA methylation arrays requested by the neuropathologist and were included in the diagnostic cohort. Molecular profiling added a unique contribution to clinical diagnosis in 107 (35%; 95% CI 30-40) of 306 cases in routine diagnostic practice-providing additional molecular subtyping data in 99 cases, amended the final diagnosis in five cases, and making potentially significant predictions in three cases. We estimated that it could change conventional management in 11 (4%; 95% CI 2-6) of 306 patients. Among 195 historically difficult-to-diagnose tumours in the archival cohort, 99 (51%) could be diagnosed using standard methods, with the addition of methylation profiling solving a further 34 (17%) cases. The remaining 62 (32%) cases were unresolved despite specialist pathology and methylation profiling. INTERPRETATION: Together, these data provide estimates of the impact that could be expected from routine implementation of genomic profiling into clinical practice, and indicate limitations where additional techniques will be required. We conclude that DNA methylation arrays are a useful diagnostic adjunct for childhood CNS tumours. FUNDING: The Brain Tumour Charity, Children with Cancer UK, Great Ormond Street Hospital Children's Charity, Olivia Hodson Cancer Fund, Cancer Research UK, and the National Institute of Health Research.

Muscle Biopsy Findings in Combination With Myositis-Specific Autoantibodies Aid Prediction of Outcomes in Juvenile Dermatomyositis.

OBJECTIVE: Juvenile dermatomyositis (DM) is a rare and severe autoimmune condition characterized by rash and proximal muscle weakness. While some patients respond to standard treatment, others do not. This study was carried out to investigate whether histopathologic findings and myositis-specific autoantibodies (MSAs) have prognostic significance in juvenile DM. METHODS: Muscle biopsy samples (n = 101) from patients in the UK Juvenile Dermatomyositis Cohort and Biomarker Study were stained, analyzed, and scored for severity of histopathologic features. In addition, autoantibodies were measured in the serum or plasma of patients (n = 90) and longitudinal clinical data were collected (median duration of follow-up 4.9 years). Long-term treatment status (on or off medication over time) was modeled using generalized estimating equations. RESULTS: Muscle biopsy scores differed according to MSA subgroup. When the effects of MSA subgroup were accounted for, increased severity of muscle histopathologic features was predictive of an increased risk of remaining on treatment over time: for the global pathology score (histopathologist's visual analog scale [hVAS] score), 1.48-fold higher odds (95% confidence interval [95% CI] 1.12-1.96; P = 0.0058), and for the total biopsy score (determined with the standardized score tool), 1.10-fold higher odds (95% CI 1.01-1.21; P = 0.038). A protective effect was identified in patients with anti-Mi-2 autoantibodies, in whom the odds of remaining on treatment were 7.06-fold lower (95% CI 1.41-35.36; P = 0.018) despite muscle biopsy scores indicating more severe disease. In patients with anti-nuclear matrix protein 2 autoantibodies, anti-transcription intermediary factor 1γ autoantibodies, or no detectable autoantibody, increased histopathologic severity alone, without adjustment for the effect of MSA subtype, was predictive of the risk of remaining on treatment: for the hVAS global pathology score, 1.61-fold higher odds (95% CI 1.16-2.22; P = 0.004), and for the total biopsy score, 1.13-fold higher odds (95% CI 1.03-1.24; P = 0.013). CONCLUSION: Histopathologic severity, in combination with MSA subtype, is predictive of the risk of remaining on treatment in patients with juvenile DM and may be useful for discussing probable treatment length with parents and patients. Understanding these associations may identify patients at greater risk of severe disease.

mTOR-dependent abnormalities in autophagy characterize human malformations of cortical development: evidence from focal cortical dysplasia and tuberous sclerosis.

Focal cortical dysplasia (FCD) is a localized malformation of cortical development and is the commonest cause of severe childhood epilepsy in surgical practice. Children with FCD are severely disabled by their epilepsy, presenting with frequent seizures early in life. The commonest form of FCD in children is characterized by the presence of an abnormal population of cells, known as balloon cells. Similar pathological changes are seen in the cortical malformations that characterize patients with tuberous sclerosis complex (TSC). However, the cellular and molecular mechanisms that underlie the malformations of FCD and TSC are not well understood. We provide evidence for a defect in autophagy in FCD and TSC. We have found that balloon cells contain vacuoles that include components of the autophagy pathway. Specifically, we show that balloon cells contain prominent lysosomes by electron microscopy, immunohistochemistry for LAMP1 and LAMP2, LysoTracker labelling and enzyme histochemistry for acid phosphatase. Furthermore, we found that balloon cells contain components of the ATG pathway and that there is cytoplasmic accumulation of the regulator of autophagy, DOR. Most importantly we found that there is abnormal accumulation of the autophagy cargo protein, p62. We show that this defect in autophagy can be, in part, reversed in vitro by inhibition of the mammalian target of rapamycin (mTOR) suggesting that abnormal activation of mTOR may contribute directly to a defect in autophagy in FCD and TSC.

Balloon cells in human cortical dysplasia and tuberous sclerosis: isolation of a pathological progenitor-like cell.

Neural stem cells are present in the human post-natal brain and are important in the development of brain tumours. However, their contribution to non-neoplastic human disease is less clear. We have tested the hypothesis that malformations of cortical development contain abnormal (pathological) stem cells. Such malformations are a major cause of epilepsy. Two of the most common malformations [focal cortical dysplasia (FCD) and cortical tubers] are characterised by the presence of a population of abnormal cells known as balloon cells. The identity of these cells is unknown but one hypothesis is that they are an abnormal stem cell that contributes to the pathogenesis of the malformation. We have characterised in tissue, and isolated in culture, an undifferentiated population of balloon cells from surgical resections of FCD and cortical tubers. We show that beta1-integrin labels a sub-population of balloon cells with a stem cell phenotype and show for the first time that these cells can be isolated in vitro. We have characterised the immunohistochemical, morphological and ultrastructural features of these cells. This is the first isolation of an abnormal cell with features of a progenitor/stem cell from a non-neoplastic disease of the brain.

The intergenic region between the Mouse Recql4 and Lrrc14 genes functions as an evolutionary conserved bidirectional promoter.

Mammalian genomes are highly complex, with neighbouring genes arranged in divergent, convergent, tandem, antisense, and interleaving fashions. Despite the vast genomic space, a substantial portion of human genes (approximately 10%) are arranged in a divergent, head-to-head fashion and controlled by bidirectional promoters. Here we define a small core bidirectional promoter that drives expression of the mouse genes Recql4, on one strand, and Lrrc14; a novel member of the LRR gene family, on the opposite strand. Regulation of Lrrc14 expression is highly complex, involving multiple promoters' and alternative splicing. Expression of this gene is predominately restricted to neural tissue during embryogenesis and is expressed in a wide range of tissues in the adult.