Activation of ADRB2/PKA Signaling Pathway Facilitates Lipid Synthesis in Meibocytes, and Beta-Blocker Glaucoma Drug Impedes PKA-Induced Lipid Synthesis by Inhibiting ADRB2.

Meibomian gland dysfunction is one of the main causes of dry eye disease and has limited therapeutic options. In this study, we investigated the biological function of the beta 2-adrenergic receptor (ADRB2)/protein kinase A (PKA) pathway in lipid synthesis and its underlying mechanisms in human meibomian gland epithelial cells (HMGECs). HMGECs were cultured in differentiation media with or without forskolin (an activator of adenylate cyclase), salbutamol (an ADRB2 agonist), or timolol (an ADRB2 antagonist) for up to 4 days. The phosphorylation of the cAMP-response element-binding protein (CREB) and the expression of peroxisome proliferator activator receptor (PPAR)γ and sterol regulatory element-binding protein (SREBP)-1 were measured by immunoblotting and quantitative PCR. Lipid synthesis was examined by LipidTOX immunostaining, AdipoRed assay, and Oil Red O staining. PKA pathway activation enhanced PPARγ expression and lipid synthesis in differentiated HMGECs. When treated with agonists of ADBR2 (upstream of the PKA signaling system), PPARγ expression and lipid synthesis were enhanced in HMGECs. The ADRB2 antagonist timolol showed the opposite effect. The activation of the ADRB2/PKA signaling pathway enhances lipid synthesis in HMGECs. These results provide a potential mechanism and therapeutic target for meibomian gland dysfunction, particularly in cases induced by beta-blocker glaucoma drugs.

The Anti-Atherosclerosis Effect of Anakinra, a Recombinant Human Interleukin-1 Receptor Antagonist, in Apolipoprotein E Knockout Mice.

Interleukin (IL)-1ß plays an important role in atherosclerosis pathogenesis. We aimed to investigate the effect of anakinra, a recombinant human IL-1 receptor antagonist, on the progression of atherosclerosis in apolipoprotein E knockout (ApoE−/−) mice. ApoE−/− mice (8-week male) were treated with saline (control), anakinra 10, 25, and 50 mg/kg, respectively (n = 10 in each group). Mice were fed a standard chow (4 weeks) followed by an atherogenic diet (35kcal% fat, 1.25% cholesterol, 12 weeks). Atheromatous plaques in ApoE−/− mice and the expression of inflammatory genes and signaling pathways in human umbilical vein endothelial cells (HUVECs), rat aortic smooth muscle cells (RAOSMCs), and 3T3-L1 adipocytes were assessed. Anakinra reduced the plaque size of the aortic arch (30.6% and 25.2% at the 25 mg/kg and 50 mg/kg doses, both p < 0.05) and serum triglyceride in ApoE−/− mice and suppressed inflammatory genes (IL-1ß and IL-6) expressions in HUVECs and RAOSMCs (all p < 0.05). In RAOSMCs, anakinra reduced metalloproteinase-9 expression in a dose-dependent manner and inhibited cell migration. Anakinra-treated mice exhibited trends of lower CD68+ macrophage infiltration in visceral fat and monocyte chemoattractant protein-1 expression was reduced in 3T3-L1 adipocytes. Anakinra could be a useful component for complementary treatment with a standard regimen to reduce the residual cardiovascular risk.

Novel CFTR Activator Cact-3 Ameliorates Ocular Surface Dysfunctions in Scopolamine-Induced Dry Eye Mice.

Cystic fibrosis transmembrane conductance regulator (CFTR) is highly expressed on the ocular epithelium and plays a pivotal role in the fluid secretion driven by chloride transport. Dry eye disease is one of the most common diseases with limited therapeutic options. In this study, a high-throughput screening was performed to identify novel CFTR activators capable of inducing chloride secretion on the ocular surface. The screening of 50,000 small molecules revealed three novel CFTR activators. Among them, the most potent CFTR activator, Cact-3 (7-(3,4-dimethoxyphenyl)-N-(4-ethoxyphenyl)pyrazolo [1,5-α]pyrimidine-2-carboxamide), produced large and sustained Cl- currents in WT-CFTR-expressing FRT cells with no alterations of ANO1 and hERG channel activity. The application of Cact-3 strongly activated CFTR in the ocular epithelia of mice and it also significantly increased CFTR-mediated Cl- transport in a primary cultured human conjunctival epithelium. Cact-3 strongly stimulated tear secretion in normal mice. In addition, Cact-3 significantly reduced ocular surface damage and the expression of proinflammatory factors, including interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in an experimental mouse model of dry eye disease. These results suggest that Cact-3, a novel CFTR activator, may be a potential development candidate for the treatment of dry eye disease.

Isorhamnetin Ameliorates Dry Eye Disease via CFTR Activation in Mice.

Dry eye disease is one of the most common diseases, with increasing prevalence in many countries, but treatment options are limited. Cystic fibrosis transmembrane conductance regulator (CFTR) is a major ion channel that facilitates fluid secretion in ocular surface epithelium and is a potential target of therapeutic agent for the treatment of dry eye disease. In this study, we performed a cell-based, high-throughput screening for the identification of novel natural products that activate CFTR and restore the aqueous deficiency in dry eye. Screening of 1000 natural products revealed isorhamnetin, a flavonol aglycone, as a novel CFTR activator. Electrophysiological studies showed that isorhamnetin significantly increased CFTR chloride current, both wild type and ∆F508-CFTR. Isorhamnetin did not alter intracellular cAMP levels and the activity of other ion channels, including ANO1, ENaC, and hERG. Notably, application of isorhamnetin on mouse ocular surface induced CFTR activation and increased tear volume. In addition, isorhamnetin significantly reduced ocular surface damage and expression of interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α in an experimental mouse model of dry eye. These data suggest that isorhamnetin may be used to treat dry eye disease.

Detection of recurrent, rare, and novel gene fusions in patients with acute leukemia using next-generation sequencing approaches.

Identification of gene fusion is an essential part in the management of patients with acute leukemia, not only for diagnosis but also in predicting the treatment outcome and selecting appropriate treatment. Adopting next-generation sequencing (NGS) technology for identification of gene fusion in patients with acute leukemia can be a good alternative to conventional tests. In the present study, the NGS RNA fusion gene panel test was applied to diagnostic samples of patients with acute leukemia to identify fusion genes more efficiently. Among 134 patients with acute leukemia, 53 gene fusions were detected in 52 patients. In addition to the recurrent gene fusions specified in the WHO diagnostic criteria, 11 rare or novel gene fusions were identified. Of those, two were gene fusions associated with Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), two were novel gene fusions, three were gene fusions with novel partner genes, and six were rare gene fusions from previous reports. We confirmed the clinical utility of the NGS test in identifying clinically significant gene fusions such as gene fusions involving KMT2A that has a large number of partners. Notably, Ph-like ALL-associated gene fusions could be easily identified despite the wide variety of genes involved. The results from the present study may contribute toward a better understanding of the genomic landscape of acute leukemia as well as patient management.

Suppression of Nrf2 attenuates adipogenesis and decreases FGF21 expression through PPAR gamma in 3T3-L1 cells.

Adipogenesis is the process of differentiation from preadipocytes to adipocytes and is orchestrated by various transcription factors, such as the peroxisome proliferator-activated receptor gamma (PPARγ) and the CCAAT-enhancer-binding protein alpha (C/EBPα). Oxidative stress is also a crucial factor in adipogenesis, and adipocyte differentiation is affected by the cellular redox status. The nuclear factor E2-related factor 2 (Nrf2), which is a basic leucine zipper (bZIP) transcription factor, acts as a regulator of cellular oxidative stress. Although several previous studies examined the function of Nrf2 in adipogenesis, their results were controversial. In this study, we investigated whether the suppression of Nrf2 in 3T3-L1 cells affected adipogenesis. We found that adipogenesis master regulator genes, such as PPARγ and C/EBPα, were downregulated during the differentiation stage in Nrf2-knockdown 3T3-L1 cells. Moreover, the fibroblast growth factor 21 (FGF21) and manganese superoxide dismutase (MnSOD) were markedly downregulated in Nrf2-knockdown 3T3-L1 cells. Taken together, the results of the present study suggest that the suppression of Nrf2 attenuates adipogenesis and decreases FGF21 expression through PPARγ in 3T3-L1 cells.

Circulating sortilin level as a potential biomarker for coronary atherosclerosis and diabetes mellitus.

CONTEXT: A previous genome-wide association study showed that a genetic variant of sortilin was associated with the risk of coronary artery disease (CAD). However, the role of circulating sortilin is still unknown. We investigated the potential role of plasma sortilin as a biomarker for CAD and diabetes mellitus. METHODS: We enrolled statin-naïve subjects with CAD (n = 31) who underwent coronary artery bypass surgery and control subjects (n = 116) who were free from CAD as evaluated by coronary CT angiography. The presence of diabetes mellitus was evaluated and plasma sortilin levels were measured with a commercial ELISA kit. RESULTS: Plasma sortilin levels were higher in subjects with CAD and subjects with diabetes mellitus than in those without CAD or diabetes mellitus. Subjects in the highest sortilin tertile group were older and had higher glucose and HbA1c levels, but lipid profiles in the three tertile groups were comparable. Multivariable logistic regression analysis revealed that sortilin levels were independently associated with CAD. In addition, the receiver operating characteristic curve analysis showed that plasma sortilin levels could identify the presence of CAD or diabetes mellitus. CONCLUSIONS: Elevated circulating sortilin levels are associated with CAD and diabetes mellitus and can be used as a biomarker of both diseases in statin-naïve subjects.

Activation of sphingosine kinase 2 by endoplasmic reticulum stress ameliorates hepatic steatosis and insulin resistance in mice.

UNLABELLED: The endoplasmic reticulum (ER) is the principal organelle in the cell for protein folding and trafficking, lipid synthesis, and cellular calcium homeostasis. Perturbation of ER function results in activation of the unfolded protein response (UPR) and is implicated in abnormal lipid biosynthesis and development of insulin resistance. In this study, we investigated whether transcription of sphingosine kinase (Sphk)2 is regulated by ER stress-mediated UPR pathways. Sphk2, a major isotype of sphingosine kinase in the liver, was transcriptionally up-regulated by tunicamycin and lipopolysaccharides. Transcriptional regulation of Sphk2 was mediated by activation of activating transcription factor (ATF)4 as demonstrated by promoter assays, immunoblotting, and small interfering RNA analyses. In primary hepatocytes, adenoviral Sphk2 expression elevated cellular sphingosine 1 phosphate (S1P) and activated protein kinase B phosphorylation, with no alteration of insulin receptor substrate phosphorylation. Hepatic overexpression of Sphk2 in mice fed a high-fat diet (HFD) led to elevated S1P and reduced ceramide, sphingomyelin, and glucosylceramide in plasma and liver. Hepatic accumulation of lipid droplets by HFD feeding was reduced by Sphk2-mediated up-regulation of fatty acid (FA) oxidizing genes and increased FA oxidation in liver. In addition, glucose intolerance and insulin resistance were ameliorated by improved hepatic insulin signaling through Sphk2 up-regulation. CONCLUSION: Sphk2 is transcriptionally up-regulated by acute ER stress through activation of ATF4 and improves perturbed hepatic glucose and FA metabolism.

Synthetic Strategies for Improving Solubility: Optimization of Novel Pyrazolo[1,5-a]pyrimidine CFTR Activator That Ameliorates Dry Eye Disease.

Dry eye disease (DED) is one of the most prevalent ocular diseases but has limited treatment options. Cystic fibrosis transmembrane conductance regulator (CFTR), a major chloride channel that stimulates fluid secretion in the ocular surface, may pave the way for new therapeutic strategies for DED. Herein, we report the optimization of Cact-3, a potent CFTR activator with poor solubility, to 16d, a potent CFTR activator with suitable solubility for eye drop formulation. Notably, 16d was well distributed in target tissues including cornea and conjunctiva with minimal systemic exposure in rabbit. Topical ocular instillation of 16d significantly enhanced tear secretion and improved corneal erosion in a mouse model of DED. In addition, 16d significantly reduced mRNA expression of pro-inflammatory cytokines including IL-1ß, IL-17, and TNF-α and MMP2 in cornea and conjunctiva of DED mice.

Upregulation of the serine palmitoyltransferase subunit SPTLC2 by endoplasmic reticulum stress inhibits the hepatic insulin response.

Endoplasmic reticulum (ER) stress is induced by various conditions, such as inflammation and the presence of excess nutrients. Abnormal accumulation of unfolded proteins leads to the activation of a collective signaling cascade, termed the unfolded protein response (UPR). ER stress is reported to perturb hepatic insulin response metabolism while promoting insulin resistance. Here, we report that ER stress regulates the de novo biosynthesis of sphingolipids via the activation of serine palmitoyltransferase (SPT), a rate-limiting enzyme involved in the de novo biosynthesis of ceramides. We found that the expression levels of Sptlc1 and Sptlc2, the major SPT subunits, were upregulated and that the cellular concentrations of ceramide and dihydroceramide were elevated by acute ER stress inducers in primary hepatocytes and HepG2 cells. Sptlc2 was upregulated and ceramide levels were elevated by tunicamycin in the livers of C57BL/6J wild-type mice. Analysis of the Sptlc2 promoter demonstrated that the transcriptional activation of Sptlc2 was mediated by the spliced form of X-box binding protein 1 (sXBP1). Liver-specific Sptlc2 transgenic mice exhibited increased ceramide levels in the liver and elevated fasting glucose levels. The insulin response was reduced by the inhibition of the phosphorylation of insulin receptor ß (IRß). Collectively, these results demonstrate that ER stress induces activation of the de novo biosynthesis of ceramide and contributes to the progression of hepatic insulin resistance via the reduced phosphorylation of IRß in hepatocytes.

The Effects of PPAR Agonists on Atherosclerosis and Nonalcoholic Fatty Liver Disease in ApoE-/-FXR-/- Mice.

BACKGROUND: Farnesoid X receptor (FXR), a bile acid-activated nuclear receptor, is a potent regulator of glucose and lipid metabolism as well as of bile acid metabolism. Previous studies have demonstrated that FXR deficiency is associated with metabolic derangements, including atherosclerosis and nonalcoholic fatty liver disease (NAFLD), but its mechanism remains unclear. In this study, we investigated the role of FXR in atherosclerosis and NAFLD and the effect of peroxisome proliferator-activated receptor (PPAR) agonists in mouse models with FXR deficiency. METHODS: En face lipid accumulation analysis, liver histology, serum levels of glucose and lipids, and mRNA expression of genes related to lipid metabolism were compared between apolipoprotein E (ApoE)-/- and ApoE-/-FXR-/- mice. The effects of PPARα and PPARγ agonists were also compared in both groups of mice. RESULTS: Compared with ApoE-/- mice, ApoE-/-FXR-/- mice showed more severe atherosclerosis, hepatic steatosis, and higher levels of serum cholesterol, low-density lipoprotein cholesterol, and triglycerides, accompanied by increased mRNA expression of FAS, ApoC2, TNFα, IL-6 (liver), ATGL, TGH, HSL, and MGL (adipocytes), and decreased mRNA expressions of CPT2 (liver) and Tfam (skeletal muscle). Treatment with a PPARα agonist, but not with a PPARγ agonist, partly reversed atherosclerosis and hepatic steatosis, and decreased plasma triglyceride levels in the ApoE-/-FXR-/- mice, in association with increased mRNA expression of CD36 and FATP and decreased expression of ApoC2 and ApoC3 (liver). CONCLUSION: Loss of FXR is associated with aggravation of atherosclerosis and hepatic steatosis in ApoE-deficient mice, which could be reversed by a PPARα agonist through induction of fatty acid uptake, ß-oxidation, and triglyceride hydrolysis.

Serial Detection of MYD88 L265P Mutation in the Aqueous Humor of a Patient with Vitreoretinal Lymphoma for Disease Monitoring.

PURPOSE: To report a case of a patient whose MYD88 mutation disappeared from the aqueous humor following treatment with intravitreal methotrexate. METHODS: A retrospective review of clinical, histopathological and imaging records. RESULTS: A 49-year-old woman presented with bilateral primary vitreoretinal lymphoma confirmed by molecular and next-generation sequencing studies on vitreous biopsy samples. Initially, the MYD88 L265P mutation was detected in aqueous samples of both eyes. Serial testing for MYD88 L265P mutations performed on aqueous samples collected at the time of the weekly intravitreal methotrexate injections showed the mutation ceased to be detected after four weekly injections in the non-vitrectomized right eye and after two weekly injections in the vitrectomized left eye. Clinical improvement accompanied the negativization of the mutation in both eyes. CONCLUSION: We present a case that demonstrates the possible utilization of serial testing for the MYD88 L265P mutation as a tool for monitoring disease course in vitreoretinal lymphoma.

Impact of maternal engrafted cytomegalovirus-specific CD8+ T cells in a patient with severe combined immunodeficiency.

OBJECTIVES: In patients with severe combined immunodeficiency (SCID), the immune system often fails to eradicate maternal cells that enter the foetus via the placenta, resulting in transplacental maternal engraftment (TME) syndrome. However, the clinical significance of TME has not been comprehensively elucidated. METHODS: Here, we describe a patient with SCID with a novel frameshift IL2RG mutation associated with maternal engrafted CD8+ T cells that had been expanded by viral infection. To evaluate the origin of the expanded T cells, we HLA-typed the myeloid and T cells of the patient and analysed the immunological characteristics of the expanded CD8+ T cells using T-cell receptor (TCR) repertoire and flow cytometry analysis. RESULTS: In our patient, the maternal engrafted CD8+ T cells expanded and exerted in vitro antiviral function against human cytomegalovirus (CMV) infection before and after haematopoietic cell transplantation (HCT). After haploidentical HCT from the maternal donor, maternal engrafted CMV-specific CD8+ T cells were maintained, successfully proliferated and activated against CMV. We found no evidence of acute graft-versus-host disease or infectious complications other than recurrent episodes of CMV viraemia, which were well controlled by ganciclovir and, possibly by, the maternal engrafted CMV-specific CD8+ T cells. CONCLUSION: Our findings elucidate a possible functional role of TME in controlling CMV infection in patient with SCID and suggest an optimal strategy for donor selection in patients with SCID with TME.

Interleukin-4 stimulates lipogenesis in meibocytes by activating the STAT6/PPARγ signaling pathway.

PURPOSE: This study was performed to investigate the effects of interleukin (IL)-4 on adipogenesis and the underlying molecular mechanisms in human meibomian gland epithelial cells (HMGECs). METHODS: HMGECs and human white preadipocytes (HWPs) were cultured and differentiated with or without IL-4. Oil-red O staining, Adipored assay, and LipidTox immunostaining were performed to examine the extent of lipid droplet formation. The expression of signal transducer and activator of transcription 6 (STAT6), phospho-STAT6, peroxisome proliferator activator receptor (PPAR)γ, and sterol regulatory element-binding protein (SREBP)-1 was measured through immunoblotting. Cells were treated with STAT6 inhibitor, which prevents the phosphorylation of STAT6 by IL-4, to determine whether the effects of IL-4 on lipogenesis were altered. RESULTS: Treatment with IL-4 significantly facilitated lipid production in differentiated HMGECs. Phosphorylation of STAT6 and expression of key adipogenesis-related molecules PPARγ and SREBP-1 were increased after IL-4 treatment. Inhibition of STAT6 phosphorylation suppressed IL-4-mediated lipid synthesis in HMGECs. In contrast, the lipid synthetic effects of IL-4 were not observed in differentiated HWPs. CONCLUSIONS: IL-4 appears to promote lipid synthesis in meibomian gland epithelial cells through the STAT6/PPARγ signaling pathway. This mechanism can serve as a potential therapeutic target for meibomian gland dysfunction.

Reanalysis of Genomic Sequencing Results in a Clinical Laboratory: Advantages and Limitations.

Genetic diagnosis of patients with neurodevelopmental disorders is imperative and a standard clinical practice. Considering the continuous accumulation of data on disease-causing variants, reanalysis of previously established sequencing data is important. Periodic reanalysis of variants with uncertain significance has become mandatory in clinical laboratories. Therefore, to confirm the utility of the reanalysis of targeted gene panel data in clinical laboratories, we re-evaluated the data of two groups of patients who had undergone targeted gene panel testing for neurodevelopmental disorders (n = 116) and epileptic encephalopathy (n = 384). This reanalysis was based on a reannotation process reflecting updated databases. Six (5.2%) and seven (1.8%) new pathogenic or likely pathogenic variants were identified in these two groups, respectively, attributable to the updated guidelines and de novo reports from unrelated patients. Although relatively low, considerable increase in the diagnostic yield was confirmed. We suggest that reanalysis of genetic variants, mainly using changes in databases and updated interpretations, should be implemented as a routine practice in clinical laboratories.

Targeted gene panel sequencing in early infantile onset developmental and epileptic encephalopathy.

BACKGROUND: Early-onset developmental and epileptic encephalopathy (DEE) is characterized by repeated seizures beginning within 3 months of birth and severe interictal epileptiform discharge, including burst suppression. This study assessed the utility of targeted gene panel sequencing in the genetic diagnosis of this disease. MATERIALS AND METHODS: Targeted gene panel sequencing was performed in 150 early infantile-onset DEE patients (≤3 months of age), and we extensively reviewed their clinical characteristics, including therapeutic efficacy, according to genotype. RESULTS: Of the early infantile-onset DEE patients, 70 were neonatal-onset DEE and the other 80 patients began experiencing seizures from 1 to 3 months after birth. There were 11 different pathogenic or likely pathogenic variants among 34.7% (52/150) of patients with early infantile-onset DEE, in whom KCNQ2, STXBP1, CDKL5, and SCN1A were the major pathogenic variants. Among the neonatal-onset DEE patients, pathological genes were identified in 42.9% (30/70), indicating a significantly higher diagnostic yield than in 27.5% (22/80) of patients who experienced seizure onset 1 to 3 months after birth (p = 0.048). Among the neonatal-onset DEE group, variants in KCNQ2, STXBP1, and CDKL5 were detected at high frequencies, accounting for 66.7% (20/30) of the pathogenic or likely pathogenic variants found in this study. CONCLUSION: Targeted gene panel sequencing demonstrated a high yield of pathogenic variants in the diagnosis of early-onset epileptic encephalopathy, especially in those with neonatal-onset DEE. Early diagnosis of early-onset epileptic encephalopathy may improve the prognosis of patients by earlier selection of appropriate treatment based on pathogenic variant.

Genetic diagnosis and clinical characteristics by etiological classification in early-onset epileptic encephalopathy with burst suppression pattern.

BACKGROUND: Early-onset epileptic encephalopathies with burst suppression (EOEE-BS) are a group of neonatal epileptic syndromes characterized by intractable epilepsy and severe psychomotor delay with structural and metabolic factors accounting for major etiologies. However, recent advances in gene sequencing have identified that genetic factors might also play a significant role in the development of EOEE-BS. Herein, we used various genetic tests to identify pathogenic genetic variants in EOEE-BS irrespective of structural malformations and analyzed the clinical features associated with each different etiology. METHODS: A total of 48 patients with EOEE-BS were included. Except for patients with severe hypoxic damage, patients with structural malformations were included in our patient cohort. Clinical features of the patients were reviewed, and etiological diagnoses were made based on several genetic tests, metabolic studies, and radiological findings. RESULT: A genetic diagnosis was made in 31 (64.6 %) patients, with the most commonly diagnosed gene being STXBP1 (n = 13, 27.1 %), followed by KCNQ2 (n = 5, 10.4 %), SCN2A (n = 5, 10.4 %), DEPDC5 (n = 3, 6.3 %), CASK (n = 1, 2.1 %), CDKL5 (n = 1, 2.1 %), GNAO1 (n = 1, 2.1 %), SLC6A8 (n = 1, 2.1 %), and LIS1 deletion (n = 1, 2.1 %). Other than the classification of epilepsy syndrome, no clinical features were associated with the genetically diagnosed group. Among eight patients with structural malformations, genetic diagnosis was achieved in five (62.5 %), and those patients had pathogenic mutations in DEPDC5 and CASK or LIS1 deletion, indicating the significance of gene sequencing irrespective of structural abnormalities. Treatment responses to a variety of medications and the ketogenic diet differed by etiology, and surgical resection proved to be effective in patients with cortical dysplasia. CONCLUSION: Genetic etiologies are an important factor in EOEE-BS irrespective of structural malformations and the treatment options may differ by etiology.

Clinical Implementation of Targeted Gene Sequencing for Malformation of Cortical Development.

BACKGROUND: Malformations of cortical development comprise phenotypically heterogeneous conditions, and the diagnostic value of genetic testing in blood still remains to be elucidated. We used targeted gene sequencing to identify malformations of cortical development caused by germline mutations and characteristics associated with pathogenic mutations. METHODS: A total of 81 patients with malformations of cortical development were included. Genomic DNA was isolated from peripheral blood. Ninety-six genes were assessed using a targeted next-generation sequencing panel. Single-nucleotide variants and exonic and chromosomal copy number variations were examined with our customized pipeline. RESULTS: Genetic causes were identified from blood in 19 (23.5%) patients with malformations of cortical development; 14 patients had pathogenic or likely pathogenic single-nucleotide variants in seven genes, including DCX (n = 5), DEPDC5 (n = 2), PAFAH1B1 (n = 3), TUBA1A (n = 1), TUBA8 (n = 1), TUBB2B (n = 1), and TUBB3 (n = 1). Five patients had pathogenic copy number variations. Multifocal involvement of the lesion (tangential distribution, P < 0.001) and concurrent involvement of multiple structures such as the cortex, white matter, and ventricle (radial distribution, P = 0.003) were more commonly found in patients with identified genetic causes. Intellectual disability was also more commonly associated with pathogenic mutations (P = 0.048). In a multivariable regression analysis, both tangential and radial radiological distribution of malformations of cortical development were independently associated with positive germline test results. CONCLUSION: We identified germline mutations in almost one-fourth of our patients with malformations of cortical development by using targeted gene sequencing. Germline abnormalities were more likely found in patients who had multifocal malformations of cortical development.