Analysis Of The Etiology And Pathogenesis Of A Family With Intellectual Disability And Development Delay

Background and purposeWe have found a family with Intellectual Disability/Developmental delay.Patients in this family have two different types of phenotypes.To investigate the causes of these two phenotypes in patients in this family,we designed relevant experiments on the possible pathogenic mechanisms of intellectual disability/stable growth,and based on the previous experimental results we analysis of the clinical phenotypes of family members.Besides,we inferred the genetic pattern of the family,selected some possible pathogenic causes for in-depth research,and analyzed their possible pathogenic mechanisms.Through model organisms to verify,we try to explain the cause of the patient’s illness,find feasible treatment methods or countermeasures,provide genetic counseling for family members,and provides valuable advice for subsequent births of members of the family,in order to ease the stress of patients and family members of the family in life.MethodsWe collected peripheral venous blood of line members in the family,extracted DNA,and collected clinical data of family members.Peripheral blood lymphocytes were used to perform karyotype analysis to analyze whether the patient’s chromosome was abnormal.Microarray comparative genomic hybridization experiments were used to perform genome-wide testing on some patients.We compared the results in the OMIM database,infered the possible cause of disease according to the literature,and selected some mutations for further research.Primers were designed for the 16 genes on the selected target fragment,and the CDS region of the most extensive transcript of human DNA of the corresponding gene was amplified and reverse-transcribed into cDNA.We cloned the above genes into the expression vector PCS2 + and synthesized mRNA in vitro.Then we introduced mRNA into zebrafish fertilized eggs in pairs by microinjection.The head size was observed and photographed at 4.25 dpf,the width of the zebrafish head was measured in a computer,and the differences between different groups were counted with software.For the group with significant differences,we introduced two of the genes into the fertilized eggs of zebrafish separately to verify whether similar effects still occur.Finally,we selected one of these genes for follow-up experiments,including overexpression and knockdown experiments to explore its effect on zebrafish head and body length,as well as experiments to explore whether this effect is related to zebrafish apoptosis and pan-neuron development.ResultsThe karyotype analysis was performed on some members of the family(patients and normal people),and the karyotype analysis results were all normal.Comparative genomic hybridization experiments found that the pioneer(Ⅳ-10)and other patients with similar symptoms(Ⅳ-14)had 2.2Mb 2q terminal deletion on chromosome 2 and 4.6Mb 4p terminal duplication on chromosome 4.On the contrary,Ⅳ-18,with a short stature,showed 2.2Mb 2q terminal duplication on chromosome 2 and 4.6Mb 4p terminal deletion on chromosome 4,Zebrafish overexpression mRNA experiments found that both the STK25 and SEPT2 injection group and the PPP1R7 and OTOS injection group had a larger zebrafish head width than the control group.PPP1R7 was selected for follow-up experiments.Compared with the control group,the overexpression of this gene did make the head of zebrafish juvenile grow larger,but after knockdown this gene,the head of zebrafish juvenile became smaller and there was no significant difference in body length.The over-expression of PPP1R7 gene apoptosis was more serious.Over-expression or knockdown of the PPP1R7 gene reduced the expression of the fluorescent reporter gene on the transgenic zebrafish embryo,that is,the development of zebrafish pan-neurons was affected.