Screening Of Schizophrenia Risk Genes And Analyses Of Behavioral Phenotypes And Mechanism Of DDHD2 Knockout Mice

Schizophrenia is a common and serious mental disorder with a prevalence rate of about 1%.It is characterized by persistent and disabling illnesses that place a heavy burden on the patient’s family and society as a whole,and has become an important problem for health care.The etiology of schizophrenia is complex,and genetic factors play an important role.The estimated heritability is about 80-85%.Studies have shown that both common and rare variants contributed to the susceptibilityof schizophrenia.At present,Genome-wide Association Study（GWAS）has found many common variants associated with schizophrenia,but the specific biological mechanism is still unclear for most of the variants;The role of rare variants has also received increasing attention,but due to their low frequency,larger sample sizes are often required.Therefore,the genetic mechanism of schizophrenia still needs a lot of in-depth research.This project aimed to analyze the role of common and rare variants in schizophrenia:1)A case-control study was conducted to analyze the association of 10 rare variant risk genes identified in the European population with schizophrenia in the Chinese Han population;2)An animal model based phenotype and mechanism research was carried out for the schizophrenia risk gene DDHD2（DDHD Domain Containing 2）implicated in our previous common variant analysis.Part I:The international Schizophrenia Exome Sequencing Meta-analysis（SCHEMA）consortium found that ultra-rare coding variants in 10 genes were significantly associated with elevated risk of schizophrenia by analyzing whole-exome sequencing data from 24,248 schizophrenia cases and 97,322 controls（predominantly European populations）.These genes have the highest expression levels in the central nervous system and have various biological functions including synapse formation.However,the association between these 10 risk genes and schizophrenia in other populations is unclear due to genetic differences between different ethnic groups.This study aimed to analyze the association between 10 risk genes screened in the European population and schizophrenia in the Chinese population.We genotyped the above 10 risk genes by custom exome microarrays in a Chinese population including 3,486schizophrenia cases and 3,350 normal controls.After quality control and VEP tool annotation,the impact of missense variants on protein function was assessed based on SIFT and Poly Phen-2 scores,58 variants with potential impact on protein function were retained,and finally association analysis was performed using the SHEsis Plus online software platform（16 variants in 7 genes were retained by excluding 42 monomorphic variants from the association analysis）.In the loci analysis results,a total of three missense variants were detected only in schizophrenia cases,including TRIO（rs145664044,p.Arg1185Gln）associated with neuronal migration and growth,the transcriptionally regulated gene RB1CC1（rs113117391,p.Arg1514Cys）,the protein ubiquitination-linked gene HERC1（p.Val4517Leu）.In gene-level association analysis,RB1CC1（OR=1.634;95%CI:1.062-2.516;p=0.025）was identified as a risk gene for schizophrenia in Chinese Han Chinese.RB1CC1 is not only involved in transcription,autophagy,and DNA damage repair,but also plays an indispensable role in regulating neuronal endostasis.This study provides a reference and basis for subsequent functional studies in schizophrenia.Considering the rarity of rare variants and the limitations of custom exon array microarrays,a more comprehensive validation analysis of all exon loci of related genes using large sample sizes is required.Part II:The previous group discovered the association between genetic polymorphisms on the 8p12 region and schizophrenia through GWAS for the first time,and clarified that the related risk loci have a regulatory role in phospholipase（DDHD2）gene expression through fine localization and functional locus analysis.The product encoded by the DDHD2 gene is one of the important members of the phospholipase A1 family,which is highly expressed in the digestive system such as the brain and gastrointestinal tract,and is involved in the maintenance of glycolipid metabolism.Studies have shown that the homeostasis of glucose and lipid metabolism in the body can affect the intestinal flora,leading to the dysfunction of the"brain-gut-microbe axis",which is involved in the occurrence and development of various psychiatric diseases.In order to clarify the mechanism of the involvement of DDHD2 gene in schizophrenia,this study mainly focused on the schizophrenia-like behavioral analysis of DDHD2 gene knockout mice,and explored the molecular mechanism of its pathogenicity by means of gut microbiota and single-cell sequencing.We constructed MK-801-induced,DDHD2-selective inhibitor,and DDHD2^-/-knockout mice and performed Open-Field,Barnes Maze,and Three-box Social behavior experiments,which showed that both DDHD2^-/-and DDHD2-selective inhibitor mice exhibited behavioral abnormalities similar to the MK-801-positive mouse model,with more pronounced anxiety behavior and social impairment compared to controls,and the DDHD2^-/-knockout mice had a more pronounced schizophrenic phenotype indicating that the DDHD2 gene can induce schizophrenia-like behavior.Analysis of fecal gut flora diversity revealed that DDHD2 knockout produced significant changes in the abundance and diversity of mouse gut microbial communities,and the predicted functional composition of microbial communities suggested that differential gut flora was closely associated with 24 functions,such as carbohydrate transport and metabolism,transcription,and signal transduction mechanisms,and defense mechanisms were elevated in DDHD2^-/-mice.Antibiotic intervention in DDHD2^-/-knockout animal models significantly improved schizophrenia-like behavior,suggesting an important role for the gut microbiota.Subsequently,a total of 25 cell types were found by single-cell sequencing of brain tissue,among which the spiral ganglion neuron region showed significant differences between DDHD2^-/-mice and wild-type mice.Further analysis of biological pathway classification and enrichment showed that three biological pathways,glutamatergic synapse,GABAergic synapse,and bile secretion,were most significantly associated with the phenotype of DDHD2^-/-mice.In this study,we found that DDHD2 knockout mice exhibited significant anxiety-like behaviors,memory loss,and reduced social interest in schizophrenia-like behavioral abnormalities,and preliminarily elucidated the intestinal flora,related cell types,and biological pathways affecting DDHD2-induced schizophrenia-like behaviors.In summary,this thesis focused on a series of studies on the role of common and rare variants in the pathogenesis of schizophrenia.The RB1CC1 gene was found to be a potential risk gene for schizophrenia in the Chinese population,the DDHD2 gene alteration was confirmed to cause schizophrenia-related behavioral abnormalities,and suggested a role of gut microbiota changes mediated by DDHD2,the specific biological mechanism of which needs to be further elucidated.