Genetic Analysis Of Oral-facial-digital Syndrome Type ? Caused By CPLANE1 Gene Mutations And Preliminary Exploration Of CPLANE1 Gene Function

Objective: Objective: This study is based on a genetic analysis of a suspected type VI family with oral and facial finger syndrome,which provides the basis for its genetic counseling and prenatal diagnosis;the effect of CPLANE1 gene on mouse embryonic fibroblasts(NIH/3t3)and its primary cilia was studied and the function of CPLANE1 gene was preliminarily discussed.Methods:1.We collect the prenatal examination results of probands in the family and ask pregnant women for detailed pregnancy and family history;2.Combined with the clinical characteristics of the probands,whole exon sequencing and bioinformatics analysis were performed on family members,and related inspections such as karyotype analysis and copy number variation were also improved;3.We performed Sanger sequencing verification on the detected suspicious disease-causing mutations,and then searched Clinvar,OMIM,HGMD and other related databases and drawn pedigree maps;4.Combining the results of sequencing and bioinformatics analysis,the pathogenic gene is considered to be the CPLANE1 gene.We designed and synthesized three CPLANE1-si RNAs and one negative control si RNA,and used liposome Lipo2000 to transfect si RNA on NIH/3t3 cells to interfere with CPLANE1 gene expression,while exploring the optimal transfection conditions(such as transfection time,transfection reagents)Ratio,cell density,etc.),and screened a CPLANE1-si RNA with the highest interference efficiency by RT-q PCR.5.After finding the most suitable transfection conditions and the si RNA with the highest interference efficiency,the experiment was divided into four groups: blank control group,Lipo2000 control group,NC-si RNA group,CPLANE1-si RNA group,using CCK-8 method to study cell activity,flow cytometry to study apoptosis and cycle,Transwell to study cell migration;6.The cilia of four groups of cells were observed by immunofluorescence staining: Acetyl K40 labeled ciliary body,?-Tubulin labeled cilia base,and DAPI labeled nuclei.Results:1.The proband is the mother's third pregnancy.Prenatal ultrasound indicated that the proband had a "Blake" cyst and polydactyly of the feet.During the first pregnancy of the proband's mother,prenatal ultrasound indicated fetal corpus callosum hypoplasia,cerebellar vermis dysplasia,and polyphasic malformation on the axis of both hands.The proband's mother had an unexplained miscarriage during her second pregnancy.The proband's mother had delivered a healthy baby in her fourth pregnancy.The couple denied that their close relatives were married and denied a family history of the disease;2.Whole exon sequencing combined with clinical phenotype found that there was a compound heterozygous mutation in the proband CPLANE1 gene,the mother carried a missense mutation in A,and the father carried a B shear mutation,Prophet's brother / sister are wild homozygous at A and B sites.The karyotype analysis and copy number variation test results of the proband and his younger brother/ sister are not abnormal;3.After Sanger sequencing verification and database query,the software analysis found that the missense mutation of CPLANE1 gene A was an unknown mutation(biased to cause disease),and the B-shear mutation was a disease-causing mutation.Therefore,the proband is considered to be type VI of oral and facial finger syndrome.Draw a family tree based on the above information;4.After searching for the si RNA with the highest interference efficiency,CPLANE1 interference efficiency can reach 90%.Use this condition for subsequent experiments;5.CCK-8 experiment shows that the cell activity of CPLANE1-si RNA group is significantly reduced compared with NC-si RNA group(p<0.01);Apoptosis experiments showed that the CPLANE1-si RNA group had increased apoptosis(p<0.05)compared to the NC-si RNA group;cell cycle experiments showed that the blank control group,Lipo2000 control group,NC-si RNA group,CPLANE1-si RNA group The proportion of cells in G1,S,and G2 phases was not significantly different(p>0.05);Transwell experiments showed that the number of cell migration in CPLANE1-si RNA group was significantly more than that in NC-si RNA transfection group(p<0.01);6.After immunofluorescence staining of cilia,about 70% to 76% of cells in the blank control group,Lipo2000 control group,and NC-si RNA group showed cilia structure,each cell had only one cilia,and the length was between 5 and10 ?m.The number of cilia in the CPLANE1-si RNA group was significantly less than that in the NC-si RNA transfection group(p<0.01).Conclusion: 1.This study found a family with suspected Oral-Finger Finger Syndrome Type VI through genetic testing and bioinformatics analysis,which provides powerful help for genetic counseling and prenatal diagnosis of the family;2.After expression,NIH / 3t3 cell activity decreased,apoptosis rate increased,migration rate decreased,but the cycle was not affected;3.Low expression of CPLANE1 can cause the number of cilia on NIH / 3t3 cells to be significantly reduced.