Phenotype And Genotype Analysis Of Cornelia De Lange Syndrome And Mechanism Of Growth Retardation Caused By NIPBL

Background and ObjectiveCornelia de Lange Syndrome(CdLS,OMIM:122470,300590,300882,610759 and 614701)encompasses a broad spectrum of phenotypes characterized by distinctive craniofacial abnormalities,limb malformations,growth retardation,and intellectual disability,which was estimated prevalence of 1.6～2.2:100,000 and no racial disparity in prevalence.Molecular investigations have attributed the etiology of CdLS to genetic variants of structural or regulatory components of the cohesin complex.Seven genes associated with CdLS have been identified,and NIPBL is the most common gene.The clinical and genetic characteristics of NIPBL were analyzed,including neonatal period,infancy,early childhood and adolescence,and the relationship between genotype and phenotype of NIPBL was expounded.The iPSC cell model of 5 individual CdLS was established for the first time in the world,and the mechanism of NIPBL gene variation on CdLS growth retardation was studied based on iPS cell.Methods1.We collected data regarding the neonatal period,maternal status,laboratory examination,clinical manifestation,including facial dimorphisms and development,and follow up treatment for individuals diagnosed with CdLS.2.Whole exome Sequencing and Sanger sequencing were performed to verify the diagnosis in individuals with suspected CdLS.Meanwhile,the clinical manifestations and gene variation reported in Chinese were analyzed by using database.3.Peripheral blood mononuclear cells(PBMC)of children with different genetic variants CdLS were isolated,and reprogrammed into iPSCs by plasmid electrotransfer technology.The pluripotent molecular markers of iPSCs were identified by cellular immunofluorescence technique.The expression of endogenous pluripotent genes,including TRA-60,TRA-81,OCT4,SSEA4 and NANOG,were detected by qRT-PCR.The expression of molecular markers in the three germ layers was detected by embryoid body self-differentiation assay to verify the multidirectional differentiation potential of iPSCs.G-band chromosome analysis was performed to detect genetic abnormality.Sanger sequencing was used to confirm the NIPBL mutation in the generated iPSCs.4.The iPS cell lines of 4 CdLS children with NIPBL variation and the normal iPS cell lines of the corresponding age and gender control were cultured normally,and the cell proliferation and multiplication differences were observed.CCK8 proliferation assay kit was used to detect the cell survival rate of patients and controls,and flow cytometry was used to detect the difference in cell cycle.5.The differences of NIPBL and CCND1 gene expression between CdLS group and the control group were detected by qRT-PCR.The changes of intracellular distribution of NIPBL and CCND1 were detected by immunofluorescence technique.The differences in NIPBL and CCND1 protein levels were detected between the two groups by Western blot.6.Wnt signal activator LiCl was used to intervene cells,and the effects on cell proliferation,cell cycle and CCND1 expression were observed obviously.Results1.Clinical phenotype:the average score of 12 cases was 13.8±1.7 according to international public recognition,and the ratio of male to female was 1:1.The prenatal growth retardation and postnatal growth retardation was 10/12(83.33%),and the postnatal growth retardation was 11/12(91.67%).Synophrys and/or thick eyebrows,long and smooth philtrum,the global developmental delay and/or intellectual disability,microcephaly,small hands,long eyelashes,high palatal arch,micrognathia,and cryptorchidism accounted for 100%in the patients with CdLS.Patients 4 and 7 were treated with growth hormone(GH)to improve their growth rate.Patients 11 had hypogonadotropin-induced hypogonadism.2.For the craniofacial features,the average score of the NIPBL gene variants was higher than that of other genes.The variation types of NIPBL were compared from the cardinal feature scores,the degree of height deviation from normal height,and the severity of disease.The nonsense and frameshift pathogenic variants were more serious than missense and splicing pathogenic variants.3.A total of 41 Chinese patients with CdLS were searched from the database,and NIPBL(83.33%)was the most common genotype.Eight clinical features of these characteristics were significantly different in Chinese population compared with those different regions,8 clinical features of these characteristics were significantly different in Chinese population,including Synophrys and/or thick eyebrows,long eyelashes,cleft palate,micromelia,crease abnormalities,gastroesophageal reflux,ptosis and hearing loss.4.Establishment and identification of iPS cell:After reprogramming,four PBMC strains derived from children carrying NIPBL gene variants and HDAC8 gene variants showed significant embryonic stem cell morphology.After reprogramming,not only pluripotency markers can be expressed,but also the typical embryoid body structure can be self-differentiated and the specific markers of inner,middle and ectoderm can be detected.The constructed iPS cell had the same chromosome karyotype as the peripheral blood samples of the children,and retained the original gene variation of the cells,without integrating plasmid genes.5.The proliferation of NIPBL mutant iPS cells was significantly reduced,and cell cycle analysis showed that the cell cycle was arrested in G0/G1 phase,which was consistent with the clinical characteristics of growth retardation.6.Compared with the control,the mRNA expression levels of NIPBL and CCND1 in iPSCs of patients were significantly decreased,Western blot analysis showed that NIPBL mutation significantly reduced the protein levels of NIPBL and CCND1.The variation of NIPBL did not affect their intracellular distribution.7.LiCl,which is Wnt signal agonist can improve the inhibition of cell cycle.After 5mMLiCl intervention,it was found that the proliferation ability of the cells was significantly improved,the inhibition of G0/G1 phase of the cell cycle was improved,and the expression level of CCND1 was significantly increased.Conclusions1.In this study,the consensus scores reported by phenotype was typical CdLS,with the typical features of craniofacial features,short hands or/and feet,growth retardation,intellectual disability,and cryptorchidism.2.For the first time,the results of growth hormone stimulation test in 3 patients with CdLS were reported,and the diagnosis of growth hormone deficiency was confirmed.At the same time,2 patients were treated with growth hormone,which proved that the application of growth hormone intervention can improve the growth rate.3.In this study,we reported the clinical and genetic characteristics of 12 Chinese children with CdLS,including 8 cases of new pathogenic variants,which expanded the genetic spectrum.4.The clinical phenotypes of nonsense and frameshift variants of NIPBL gene variants are more serious than splicing and missense variants.NIPBL was the most common genotype in Chinese children with CdLS.The phenotype of Chinese population was similar to that of other countries and regions,and eight significant clinical features were found.5.The iPS cell for PBMC-derived CdLS was successfully constructed by nonintegrated reprogramming technology for the first time,and the iPS cell model for CdLS was established.6.Based on iPS cell,the cells were found to proliferate slowly.It was proved that the expression levels of mRNA and protein were decreased due to NIPBL gene variation,thus Wnt pathway was blocked,cell cycle was arrested and cell proliferation was decreased,which was consistent with the clinical characteristics of growth retardation.7.The application of LiCl,which is an activator of Wnt signaling,can improve cell proliferation,which provides a new idea for studying the mechanism of CdLS growth retarding and a cytological research model for the next step of drug screening.