The Screening Of Core Family Affected With Autism Spectrum Disorder Via Whole Exome Sequencing And Effect On Behavior In Early Postnatal Fluoxetine Treated On Zebrafish

Part 1 Identification genetic cause on the childhood disintegrative disorder by whole exome sequencingObjectivesChildhood disintegrative disorder(CDD),which is known as a subtype of Autism Spectrum Disorders,is a rare progressive neurologic disorder.However the pathogenesis and etiology of CDD is not clear.Two brothers with the same regression pattern who both lost ability in language and life skills gradually since 4 years old,were diagnosed strictly with CDD by experienced child psychiatrist.The aim of our study is trying to explore the possible etiology of CDD from the siblings.MethodsWhole exome sequencing was first used to screen the whole family members including the healthy parents;clinical scales including Autism Diagnostic InterviewRevised,the Child Autism Rating Scale score and Autism behavior checklist were applied to assess their clinical features;Further MRI scan was performed to exam the families' brain structure and function;Finally,in order to vertify the genetic results,we added the biochemical and metabolic detection including an uGAG assay and enzymes activity test.ResultsThere was a new mutation p.F101S(c.302T>C)in the gene SGSH fits the homozygous recessive genetic model: the siblings were homozygous mutant and their parents are heterozygous carriers.By MRI scanning,we found cerebral atrophy were general: gray matter became thinner with expanded sulcus;the white matter exhibited degenerated,especially in corpus callosum.The score of ADI-R showed the social interaction and verbal communication were far surpass the cutoff score,but repetitive behavior were relatively mild;meanwhile the scores of CARS and ABC also indicated both of probands met the criterion of severe autistic symptom,even the score of ABC were much higher than two years ago.Excess GAGs was noted in urine and only the activity of N-sulfoglucosamine sulfohydrolase was much lowerwhile the others were normal.ConclusionsWe report the new mutation F101 S in disease-causing gene SGSH in the siblings with CDD.SGSH encodes for N-sulfoglucosamine sulfohydrolase,and mutations in SGSH result in impaired degradation of heparan sulfate,classically cause Mucopolysaccharidosis ?A,a rare autosomal recessive inherited metabolic disease.The siblings' neurodegeneration symptoms were highly possible due to undegraded heparan sulfate accumulation in central nervous system.Thus,this case offered a possible etiology of CDD.Furthermore,child psychiatrists should be aware of these metabolic diseases,particularly when a patient is coupled with highly inherited tendency,or the specific regressive pattern.A genetic investigation and biochemical test should be considered when we want to clarify diagnoses and nosogenesis which importance have been proved in this study.Part 2 Indentification MECP2 mutations in 120 core family affected with Autism Spectrum Disorder based on whole exome sequencing and preliminary research of mutation functionObjectives Methyl Cp G binding protein-2(Me CP2)is an essential epigenetic regulator in human brain development.Either loss or gain of function leads to severe neurodevelopmental disorders,including Rett syndrome(RTT)and Autism Spectrum Disorder(ASD).We screened mutations of MECP2 in ASD patients via WES,further more,attempted to explore the potential function of the mutations based on molecular biology test.Methods DNA was extracted from 120 core family affected with ASD,then sequenced patients' DNA by whole exome sequencing.Which familes detected mutation in MECP2 gene were picked up for Sanger sequencing.Mouse cortical neurons and HEK-293 cells were cultured and transfected with MECP2 wild-type(WT)or mutant forms plasmid via electroporation.After 3 days in vitro,the cells were collected for western blot test and fixed for immunofluorescence to analysis mainly neurites and axon outgrowth.Results Three mutations of MECP2 were detected based on sequencing and analysis: p.P152L(c.455C>T)and p.P376S(c.1162C>T)were nonsynonymous mutations,and p.R294X(c.880C>T)was truncating mutation.Only p.P152 L was not reported before;p.P152 L and p.R294 X were de novo mutations,however,p.P376 S was inherited from proband's mother.Me CP2 overexpression in mouse cortical neurons show different influence on dendrite and axon outgrowth,between MECP2 WT and mutant forms in vitro,specifically WT and p.R294 X inhibited outgrowth of dendrite and axon,and opposite result was observed in p.P152 L,p.P376 S.Conclusions Our preliminary research to explore the potential function of MECP2 mutations indicted that MECP2 mutations disturbed functions associated with neurodevelopment,indirectly influenced the lenghth of neurites.Otherwise,these mutations have been reported in RTT.Interestingly,patients with the same mutation of MECP2 shows a broad array of clinical symptom and were diagnosed with different neurodevelopmental disorders.Therefore this study suggest the vital role of Me CP2 and it is necessary to scan the MECP2 in ASD patient,despite the frequency of mutation is not such high.Part 3 Possibly negative effect on long-lasting social-related behavior in early postnatal fluoxetine treated on zebrafishObjectives To expolore the effects to serotonin level,spontaneous movement,anxiety-like behaviour and long-term social-related behaviour via a transient early postnatal fluoxetineexposure on zebarafish larves.Methods The level of neurotransmitters and their respective metabolites in whole brain of larves(7 days post fertilization,7dpf)exposure to fluoxetine(3-6dpf)were quantified by HPLC.The effects offluoxetine on thigmotaxis behaviors as an index of anxiety-like behavior of the zebrafish larvae during the light-dark challenge phase(7-9dpf).Under the dark period(light-off),high concentrations of fluoxetine(16?M)reduced thigmotaxis of the larvae compareing with control and low concentrations on7-9dpf,particularly,the difference was statistically significant on7 dpf and 9dpf(P <0.05).Then we tested the long-term social-related behaviour on zebrafish(30dpf),quantified by social preference test.Results The level of 5-HIAA increased by enhancing dosage of fluoxetine,while the level of 5-HT,DA,DOPAC increased in low does and deceased in high dose.The 7dpf larvae s demonstrated decreased swimming distances at different fluoxetine concentrations compared with control larvaes.Low dose group(1.6 mu M,4 microns)showed less thigmotaxis than control(P <0.05)under continuous light on 7dpf?In social preference test,compared to the control group,the 4?M groupand 8?M group swam more distance in the area adjacent to the fish school(P <0.01);compared to the control group,the 4?M group and 8?M group had more time to swim in the area adjacent to the fish school(P<0.05).Conclusions The early-life exposed zebrafish showed the spontaneous movement was inhibited,the low doses of fluoxetine may be anxiolytic based on less thigmotaxis.The data from HPLC indicted DA and 5-HT system were changed by fluoxetine.Notablely,the impaired long-term social-related behavior was tested in early-life exposed zebrafish,and it was possibly caused by changed level or function of the 5-HT system which may lead to sustained effect on neural development.