A Study On Metabolic Characteristics And Mechanisms Of Intellectual Disability And KCNQ2-related Nervous System Diseases In Children

Objective: Intellectual disability(ID)is a group of neurodevelopmental disorders with complex etiology,heterogeneity of clinical manifestations,high disability rate,unclear pathogenesis,and lack of effective treatment.Therefore,early identification and intervention are of great significance for improving the prognosis of children with Global developmental delay(GDD)/ID.The aim of this study is to identify the differential metabolites in children with unexplained GDD/ID and KCNQ2-related neurological diseases,and to explore the significance of differential metabolites in the early identification of GDD/ID at the omics level,and to provide new targets for early identification and early intervention.Methods:(1)More than 1000 children with unexplained GDD/ID(blood amino acid and acylcarnitine profiles were analyzed in 1087 patients,and urinary organic acid profiles were analyzed in 1253 patients)and 100 Typically developing(TD)children who were admitted to the Department of Pediatrics,Xiangya Hospital,Central South University from October 2015 to January 2021 were enrolled and divided into GDD group,ID group and TD group.Blood and urine metabolic screening was performed by high performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry.Unsupervised Principal Component Analysis(PCA)and Orthogonal Projections to Latent structure-discriminant Analysis(OLPS-DA)were used to analyze the detection results.VIP>1.0 and P<0.05 were used as the criteria to screen differential metabolites.(2)A total of 30 patients with unexplained GDD/ID and 30 TD children who were admitted to the Department of Pediatrics,Xiangya Hospital,Central South University from March 2021 to August 2021 were enrolled,and 15 patients diagnosed with KCNQ2-related pediatric neurological diseases were recalled.After fasting blood collection in the morning,the plasma was packaged and stored for non-target metabolomics and full quantitative lipidomics detection.(3)According to the distribution of eight KCNQ2 variants in the Kv7.2 protein domain,they were divided into three groups: group A(intracellular N-terminal voltage-gated S1-S4 transmembrane region),group B(S5-S6 pore region)and group C(helix A-intracellular C-terminal region).HEK293 and CHO-K1 cell models of KCNQ2wild-type(WT)and eight variants were constructed.Real-time fluorescent quantitative PCR,Western blot,flow cytometry,enzyme-linked immunosorbent assay,immunofluorescence and other experimental techniques were used to detect the ceramide expression in KCNQ2 WT and mutants cell models.The effects of ceramide on cell proliferation,cell cycle,apoptosis,and organelle mitochondria,endoplasmic reticulum in KCNQ2 WT and mutants cell models were explored.Results:(1)Blood amino acid and acylcarnitine profiles were analyzed in 1087 patients,2.02% of the patients had clear etiology.Urinary organic acid metabolism analysis was performed in 1253 patients,1.83% of the patients had clear etiology.Twenty-eight differential metabolites of blood amino acids and acylcarnitines and 24 differential metabolites of urinary organic acids were screened out in the GDD group,while 27 differential metabolites of blood amino acids and acylcarnitines and 25 differential metabolites of urinary organic acids were screened out in the ID group.(2)Through metabolomics combined with lipidomics detection,108 small molecular differential metabolites and 48 differential lipids were found in GDD/ID patients.At the same time,98 small molecule differential metabolites and 35 differential lipids were found in patients with KCNQ2-related neurological diseases.(3)KCNQ2 variants in the intracellular N-terminal-voltage-gated S1-S4 transmembrane region group,S5-S6 pore region group and helix A-intracellular C-terminus group are pathogenic,which can affect the expression of KCNQ2 mRNA and protein.Three groups of KCNQ2 variants can cause abnormal ceramide metabolism in HEK293 cells.Ceramide can cause mitochondrial dysfunction and endoplasmic reticulum stress,and promote cell apoptosis in the three groups of KCNQ2 variant overexpressed cells.Among them,the KCNQ2 variant in the helix A-intracellular C-terminal group promotes cell apoptosis more significantly.Conclusion:(1)Blood and urine metabolic screening can be used as a routine examination method to find the etiology of GDD/ID,which can help some GDD/ID patients identify the etiology and obtain effective treatment.(2)Metabolomics combined with lipidomics can reveal the metabolic characteristics of GDD/ID and KCNQ2-related neurological diseases in children.(3)Abnormalities in sphingolipid metabolism may be related to the pathogenesis of GDD/ID.(4)Mitochondrial dysfunction and endoplasmic reticulum stress caused by ceramide may be one of the pathogenic mechanisms of KCNQ2-related neurological diseases in children.