A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Adrenomedullin2 stimulates progression of thyroid cancer in mice and humans under nutrient excess conditions.

Thyroid cancer is associated with genetic alterations, e.g. BRAFV600E , which may cause carcinomatous changes in hormone-secreting epithelial cells. Epidemiological studies have shown that overnutrition is related to the development and progression of cancer. In this study, we attempted to identify the cell nonautonomous factor responsible for the progression of BRAFV600E thyroid cancer under overnutrition conditions. We developed a mouse model for inducible thyrocyte-specific activation of BRAFV600E , which showed features similar to those of human papillary thyroid cancer. LSL-BrafV600E ;TgCreERT2 showed thyroid tumour development in the entire thyroid, and the tumour showed more abnormal cellular features with mitochondrial abnormalities in mice fed a high-fat diet (HFD). Transcriptomics revealed that adrenomedullin2 (Adm2) was increased in LSL-BrafV600E ;TgCreERT2 mice fed HFD. ADM2 was upregulated on the addition of a mitochondrial complex I inhibitor or palmitic acid with integrated stress response (ISR) in cancer cells. ADM2 stimulated protein kinase A and extracellular signal-regulated kinase in vitro. The knockdown of ADM2 suppressed the proliferation and migration of thyroid cancer cells. We searched The Cancer Genome Atlas and Genotype-Tissue Expression databases and found that increased ADM2 expression was associated with ISR and poor overall survival. Consistently, upregulated ADM2 expression in tumour cells and circulating ADM2 molecules were associated with aggressive clinicopathological parameters, including body mass index, in thyroid cancer patients. Collectively, we identified that ADM2 is released from cancer cells under mitochondrial stress resulting from overnutrition and acts as a secretory factor determining the progressive properties of thyroid cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

Non-Cell Autonomous Epileptogenesis in Focal Cortical Dysplasia.

OBJECTIVE: Low-level somatic mosaicism in the brain has been shown to be a major genetic cause of intractable focal epilepsy. However, how a relatively few mutation-carrying neurons are able to induce epileptogenesis at the local network level remains poorly understood. METHODS: To probe the origin of epileptogenesis, we measured the excitability of neurons with MTOR mutation and nearby nonmutated neurons recorded by whole-cell patch-clamp and array-based electrodes comparing the topographic distribution of mutation. Computational simulation is used to understand neural network-level changes based on electrophysiological properties. To examine the underlying mechanism, we measured inhibitory and excitatory synaptic inputs in mutated neurons and nearby neurons by electrophysiological and histological methods using the mouse model and postoperative human brain tissue for cortical dysplasia. To explain non-cell-autonomous hyperexcitability, an inhibitor of adenosine kinase was injected into mice to enhance adenosine signaling and to mitigate hyperactivity of nearby nonmutated neurons. RESULTS: We generated mice with a low-level somatic mutation in MTOR presenting spontaneous seizures. The seizure-triggering hyperexcitability originated from nonmutated neurons near mutation-carrying neurons, which proved to be less excitable than nonmutated neurons. Interestingly, the net balance between excitatory and inhibitory synaptic inputs onto mutated neurons remained unchanged. Additionally, we found that inhibition of adenosine kinase, which affects adenosine metabolism and neuronal excitability, reduced the hyperexcitability of nonmutated neurons. INTERPRETATION: This study shows that neurons carrying somatic mutations in MTOR lead to focal epileptogenesis via non-cell-autonomous hyperexcitability of nearby nonmutated neurons. ANN NEUROL 2021;90:285-299.

Incidence of cancer after asthma development: 2 independent population-based cohort studies.

BACKGROUND: Evidence regarding the risk of cancer development after asthma diagnosis is controversial and inconclusive. OBJECTIVE: This study sought to determine whether asthma is associated with an increased risk for incident cancer. METHODS: Two independent, population-based, longitudinal cohorts were examined, and estimated hazard ratios were determined using Cox regression. One group consisted of an unmatched cohort of 475,197 participants and a propensity score-matched cohort of 75,307 participants from the National Health Insurance Service-National Sample Cohort (NHIS-NSC; claims-based data from 2003 to 2015). The other group consisted of 5,440 participants from the Ansan-Ansung cohort (interview-based data from 2001 to 2014). RESULTS: The NHIS-NSC matched cohort had 572,740 person-years of follow-up, 6,885 people with new asthma diagnoses, and 68,422 people without asthma diagnoses. Adults with asthma had a 75% greater risk of incident cancer overall. The excess risk for incident cancer was greatest during the first 2 years after asthma diagnosis, and this risk remained elevated throughout follow-up. Patients with nonatopic asthma had a greater risk of overall cancer than those with atopic asthma. A high cumulative dose of inhaled corticosteroids among asthma patients was associated with a 56% reduced risk of lung cancer, but had no effect on the risk of overall cancer. The results from the NHIS-NSC unmatched cohort and the Ansan-Ansung cohort were similar to the primary results from the NHIS-NSC matched cohort. CONCLUSIONS: Asthma development was associated with an increased risk of subsequent cancer in 2 different Korean cohorts. Our findings provide an improved understanding of the pathogenesis of asthma and its relationship with carcinogenesis and suggest that clinicians should be aware of the higher risk of incident cancer among patients with asthma.

Growth Differentiation Factor 15 is a Cancer Cell-Induced Mitokine That Primes Thyroid Cancer Cells for Invasiveness.

Background: Mitochondrial stress is known to activate the mitochondrial unfolded protein response (UPRmt). The UPRmt results in the secretion of mitochondrial cytokines (mitokines), which can promote a hormetic response cell nonautonomously, and has been reported to be protumorigenic. Growth differentiation factor 15 (GDF15) is a well-characterized mitokine, which is reported to have a mitohormetic effect. Thus, we investigated whether GDF15 induction could prime a subpopulation of thyroid cancer cells to provide invasive advantages. Methods: The UPRmt, including mitokine expression, was assessed in the context of thyroid cancer in vitro and in vivo. GDF15 expression in 266 patients with papillary thyroid carcinoma (PTC) was determined by immunohistochemistry. The serum levels of GDF15 were measured in healthy subjects and PTC patients. In addition, our own and The Cancer Genome Atlas data were analyzed to determine the expression level of GDF15 in thyroid cancers. The role of GDF15 in tumor aggressiveness was investigated by observing the effects of GDF15 knockdown in BCPAP, TPC-1, 8505C, and FRO cells. Results: Pharmacological inhibition of mitochondrial oxidative phosphorylation function in thyroid cancer cells robustly increased GDF15 expression. The expression of GDF15 was associated with activation of the mitochondrial integrated stress response pathway in PTC patients. Circulating GDF15 levels were significantly higher in PTC patients than in the controls, and tumor expression of GDF15 was related to tumor aggressiveness. In vitro and in vivo knockdown of GDF15 in a thyroid cancer model showed decreased viability, migration, and invasion compared with the control cells via regulation of STAT3. Conclusions: In this study, we demonstrated that GDF15 is a mitokine induced in thyroid cancer cells upon mitochondrial stress. GDF15-induced STAT3 activation determined tumor progression in thyroid cancer. The GDF15-STAT3 signaling axis may be a target in aggressiveness of thyroid cancer.

Association between mental illness and COVID-19 susceptibility and clinical outcomes in South Korea: a nationwide cohort study.

BACKGROUND: Evidence for the associations between mental illness and the likelihood of a positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test result and the clinical outcomes of COVID-19 is scarce. We aimed to investigate these associations with data from a national register in South Korea. METHODS: A nationwide cohort study with propensity score matching was done in South Korea using data collected from the Health Insurance Review and Assessment Service of Korea. We defined mental illness as present if one of the relevant ICD-10 codes was recorded at least twice within 1 year for an outpatient or inpatient. Severe mental illness was considered as non-affective or affective disorders with psychotic features. We included all patients aged older than 20 years who were tested for SARS-CoV-2 through services facilitated by the Korea Centers for Disease Control and Prevention, the Health Insurance Review and Assessment Service of Korea, and the Ministry of Health and Welfare, South Korea. We investigated the primary outcome (SARS-CoV-2 test positivity) in the entire cohort and the secondary outcomes (severe clinical outcomes of COVID-19: death, admission to the intensive care unit, or invasive ventilation) among those who tested positive. FINDINGS: Between Jan 1 and May 15, 2020, 216 418 people were tested for SARS-CoV-2, of whom 7160 (3·3%) tested positive. In the entire cohort with propensity score matching, 1391 (3·0%) of 47 058 patients without a mental illness tested positive for SARS-CoV-2, compared with 1383 (2·9%) of 48 058 with a mental illness (adjusted odds ratio [OR] 1·00, 95% CI 0·93-1·08). Among the patients who tested positive for SARS-CoV-2, after propensity score matching, 109 (8·3%) of 1320 patients without a mental illness had severe clinical outcomes of COVID-19 compared with 128 (9·7%) of 1320 with a mental illness (adjusted OR 1·27, 95% CI 1·01-1·66). INTERPRETATION: Diagnosis of a mental illness was not associated with increased likelihood of testing positive for SARS-CoV-2. Patients with a severe mental illness had a slightly higher risk for severe clinical outcomes of COVID-19 than patients without a history of mental illness. Clinicians treating patients with COVID-19 should be aware of the risk associated with pre-existing mental illness. FUNDING: National Research Foundation of Korea.

Precise detection of low-level somatic mutation in resected epilepsy brain tissue.

Low-level somatic mutations have been shown to be the major genetic etiology of intractable epilepsy. The extents thereof, however, have yet to be systematically and accurately explored in a large cohort of resected epilepsy brain tissues. Moreover, clinically useful and precise analysis tools for detecting low-level somatic mutations from unmatched formalin-fixed paraffin-embedded (FFPE) brain samples, the most clinically relevant samples, are still lacking. In total, 446 tissues samples from 232 intractable epilepsy patients with various brain pathologies were analyzed using deep sequencing (average read depth, 1112x) of known epilepsy-related genes (up to 28 genes) followed by confirmatory site-specific amplicon sequencing. Pathogenic mutations were discovered in 31.9% (74 of 232) of the resected epilepsy brain tissues and were recurrently found in only eight major focal epilepsy genes, including AKT3, DEPDC5, MTOR, PIK3CA, TSC1, TSC2, SCL35A2, and BRAF. Somatic mutations, two-hit mutations, and germline mutations accounted for 22.0% (51), 0.9% (2), and 9.1% (21) of the patients with intractable epilepsy, respectively. The majority of pathogenic somatic mutations (62.3%, 33 of 53) had a low variant allelic frequency of less than 5%. The use of deep sequencing replicates in the eight major focal epilepsy genes robustly increased PPVs to 50-100% and sensitivities to 71-100%. In an independent FCDII cohort of only unmatched FFPE brain tissues, deep sequencing replicates in the eight major focal epilepsy genes identified pathogenic somatic mutations in 33.3% (5 of 15) of FCDII individuals (similar to the genetic detecting rate in the entire FCDII cohort) without any false-positive calls. Deep sequencing replicates of major focal epilepsy genes in unmatched FFPE brain tissues can be used to accurately and efficiently detect low-level somatic mutations, thereby improving overall patient care by enriching genetic counseling and informing treatment decisions.

Brain Somatic Mutations in Epileptic Disorders.

During the cortical development, cells in the brain acquire somatic mutations that can be implicated in various neurodevelopmental disorders. There is increasing evidence that brain somatic mutations lead to sporadic form of epileptic disorders with previously unknown etiology. In particular, malformation of cortical developments (MCD), ganglioglioma (GG) associated with intractable epilepsy and non-lesional focal epilepsy (NLFE) are known to be attributable to brain somatic mutations in mTOR pathway genes and others. In order to identify such somatic mutations presenting as low-level in epileptic brain tissues, the mutated cells should be enriched and sequenced with high-depth coverage. Nevertheless, there are a lot of technical limitations to accurately detect low-level of somatic mutations. Also, it is important to validate whether identified somatic mutations are truly causative for epileptic seizures or not. Furthermore, it will be necessary to understand the molecular mechanism of how brain somatic mutations disturb neuronal circuitry since epilepsy is a typical example of neural network disorder. In this review, we overview current genetic techniques and experimental tools in neuroscience that can address the existence and significance of brain somatic mutations in epileptic disorders as well as their effect on neuronal circuitry.

BRAF somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors.

Pediatric brain tumors are highly associated with epileptic seizures1. However, their epileptogenic mechanisms remain unclear. Here, we show that the oncogenic BRAF somatic mutation p.Val600Glu (V600E) in developing neurons underlies intrinsic epileptogenicity in ganglioglioma, one of the leading causes of intractable epilepsy2. To do so, we developed a mouse model harboring the BRAFV600E somatic mutation during early brain development to reflect the most frequent mutation, as well as the origin and timing thereof. Therein, the BRAFV600E mutation arising in progenitor cells during brain development led to the acquisition of intrinsic epileptogenic properties in neuronal lineage cells, whereas tumorigenic properties were attributed to high proliferation of glial lineage cells. RNA sequencing analysis of patient brain tissues with the mutation revealed that BRAFV600E-induced epileptogenesis is mediated by RE1-silencing transcription factor (REST), which is a regulator of ion channels and neurotransmitter receptors associated with epilepsy. Moreover, we found that seizures in mice were significantly alleviated by an FDA-approved BRAFV600E inhibitor, vemurafenib, as well as various genetic inhibitions of Rest. Accordingly, this study provides direct evidence of a BRAF somatic mutation contributing to the intrinsic epileptogenicity in pediatric brain tumors and suggests that BRAF and REST could be treatment targets for intractable epilepsy.