Genetic And Functional Mechanisms Of GIGYF1 In ASD Risk

Background:Autism spectrum disorder（ASD）is a neurodevelopmental disorder characterized by deficits in social interaction and communication,and limited and/or repetitive patterns of behavior and thought,interests,or activities.ASD has high heritability and high genetic heterogeneity.More than 200 high-confidence ASD risk genes have been identified by analyzing large cohorts of de novo or extremely rare genetic variants.Because these risk variants are extremely rare,the mutational characteristics and inheritance patterns and detailed genotype-phenotype relationships of the vast majority of ASD risk genes in the disease remain unknown.GRB10 Interacting GYF Protein 1（GIGYF1）encodes a protein involved in the regulation of insulin-like growth factor receptor signaling pathway.Previous studies have found that the loss-of-function（Lo F）de novo variant of GIGYF1 in ASD patients is significantly higher than the probability of random de novo mutations in the population,proving that de novo GIGYF1 Lo F variants are associated with the risk of ASD related.Nonetheless,the comprehensive mutational characterization and precise inheritance pattern of GIGYF1 in ASD pedigrees,as well as the detailed genotype-phenotype relationships,remain unknown.In addition,GIGYF1is an important ASD risk gene whose biological mechanisms involved in neurodevelopment and ASD behavior are completely unknown.Objective:We combined human genetics,conditional knockout mouse models and a variety of molecular biology methods to explore the genetic mechanism,genotype-phenotype relationship and the neurobiological and molecular mechanisms behind GIGYF1 in ASD and related neurodevelopmental disorders.To provide a scientific basis for early risk warning and development of possible treatment options for ASD patients with GIGYF1 risk variants.Methods:By analyzing whole-exome sequencing and whole-genome sequencing data from a large cohort,we screened out GIGYF1 Lo F variants in ASD families or patients and analyzed their mutational characteristics,inheritance patterns,and genotype-phenotype relationships.By constructing Gigyf1 nervous system conditional knockout（Nestin-Cre）mice,we explored the effect of Gigyf1 defect on mouse behavior.The expression of Gigyf1 in different developmental stages of mice and its effect on early embryonic neurogenesis were analyzed by western blotting and immunofluorescence experiments.By constructing GIGYF1 knockout（KO）cell lines and using in vitro and in vivo biochemical and molecular biology methods to explore the molecular mechanism of GIGYF1involved in neurodevelopment and ASD.Results:We identified 46 heterozygous GIGYF1 Lo F variants in35,700 patients with ASD,with 1.8-fold more variants inherited from parents than de novo variants.Both de novo mutation enrichment analysis(P<2.7×10^-12)and transmission disequilibrium analysis(P<1×10^-5)indicated that Lo F variants in this gene were significantly associated with ASD risk.The mutation rate of heterozygous GIGYF1 Lo F variants ranks second among known high-confidence ASD risk genes.It is worth emphasizing that we found an identical recurrent variant c.333del:p.L111Rfs*234 in 23 patients.This recurrent variant accounts for50%of all heterozygous GIGYF1 Lo F variants in.We found that ASD patients with GIGYF1 Lo F variants had lower rates of cognitive impairment than those with Lo F variants in other ASD high-confidence genes(P=8.5 x 10^-24).In neurological conditional knockout mice,Gigyf1haploinsufficiency resulted in ASD-like behaviors such as social impairment and anxiety,but no significant cognitive impairment.In contrast,homozygous mice developed more severe social and cognitive impairments.Haploinsufficiency or complete knockdown of Gigyf1 in the nervous system results in decreased superficial cortical neurons in the cerebral cortex,decreased neural progenitor cell（NPC）proliferation,and prolonged S phase of NPCs.Molecular mechanism studies have found that GIGYF1 knockout in HEK293T cells leads to an obstacle to the recycling of intracellular insulin-like growth factor 1 receptor（IGF-1R）,resulting in a decrease in the level of IGF-1R on the cell membrane surface.IGF-1R/ERK signaling pathway activity.At the same time,the activity of p Erk in mouse cerebral cortex NPCs also decreased.In vitro culture of NPCs from Gigyf1 conditional knockout mice further verified the reduced NPC proliferation phenotype,and that human insulin-like growth factor 1（IGF-1）could rescue this proliferation phenotype.Conclusion:Both inherited and de novo heterozygous GIGYF1 Lo F variants significantly increase the risk of ASD.Patients with heterozygous GIGYF1 Lo F variants in have a lower incidence of cognitive impairment.GIGYF1 regulates the cycling of IGF-1R on the cell surface.Heterozygous Lo F variants or haploinsufficiency of GIGYF1/Gigyf1 may contribute to early neurogenesis abnormalities and ASD or ASD-like behaviors by affecting the IGR-1R/ERK signaling pathway.46 figures,11 tables and 96 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