The Role And Molecular Mechanism Of KANSL1 Gene In The Pathogenesis Of Developmental Dysplasia Of The Hip

Objective : Developmental dysplasia of the hip(DDH)is a common bone and joint deformity in children.Delayed treatment can cause osteoarthritis symptoms such as hip pain and claudication,which affect quality of life and eventually requiring hip replacement surgery.Early detection and prevention can greatly improve the therapeutic effect of DDH.If specific genes are identified,combined with genetic diagnosis,DDH can be accurately detected in early stage and new treatments can be provided.Therefore,although scholars have already agreed that DDH is a disease of multi-gene interaction,they are still keen on the study of related genes.Our research group has carried out a fine mapping study on the 17q21 region of 103 Chinese DDH families,and for the first time,the DDH susceptibility gene in the Chinese population was located within the 11.7c M region between 17q21.31-17 q.22(D17S855-D17S790).In the early stage of this project,a family with 14 people in 3 generations and 4 people with DDH was studied in depth.Whole exome sequencing was performed on four of these patients,and only one rare heterozygous missense mutation of KANSL1 gene(c.C767T;P.s.256f)was found in the susceptible region of chromosome 17 that we had previously located is shared by three patients.Therefore,it is strongly supported that KANSL1 gene is the pathogenic gene of DDH.The KANSL1 gene,also known as KIAA1267,CENP-36,MSL1V1 or NSL1,is located on chromosome 17 and its microdeletion or mutation can cause Kd VS(Koolende Vries Syndrome).Kd VS is characterized by special facial features,low muscle tone,low cognitive and language abilities,and some patients have hip dysplasia.In the study of Kd VS,only the occurrence of hip dysplasia was mentioned,but the pathogenesis was not studied in detail.At present,there are still no other studies on the correlation between KANSL1 gene and bone and joint diseases.This study aims to determine the genetic contribution of KANSL1 gene mutation to DDH in Han population through exon sequencing of KANSL1 gene in sporadic cases of DDH combined with previous DDH family studies.Next,the changes of chondrocytes in vivo and mouse hip cartilage in vitro were observed after low expressing KANSL1 gene,to confirm whether KANSL1 gene was involved in the development of hip cartilage.Finally,introduced KANSL1 gene mutation detected in DDH family into embryonic stem cells and mice to observe the effect of the mutation on the induction of differentiation of embryonic stem cells into chondrocytes and the development of hip cartilage in mice,then analyze the mechanism of KANSL1 gene mutation leading to DDH susceptibility.methods: This study is divided into two partsPart Ⅰ: Genetic contribution of KANSL1 gene mutation in the pathogenesis of DDH in Han population and the effects following down-regulated KANSL1 gene expression on chondrocytes and hip cartilage development.1.In order to further confirm the pathogenicity of KANSL1 gene mutation,peripheral blood of 200 patients with sporadic DDH and 400 healthy controls were used for exon sequencing of all the coding sequence and cut sequence of KANSL1 gene to detect the mutation site of KANSL1 gene in the population with sporadic DDH.Combined with the mutation sites detected in DDH families in the previous stage,the genetic contribution of KANSL1 gene mutation to DDH in Han population was analyzed.2.Due to the lack of relevant studies of KANSL1 gene on bone and joint diseases,we performed lentivirus transfection into primary chondrocytes and perihip injection of lentivirus with low expression of KANSL1 gene to analyze whether KANSL1 gene was involved in hip cartilage development.KANSL1 sg RNA lentivirus constructed by Crisp/cas9 was transfected into primary chondrocytes to reduce KANSL1 gene expression.Rt PCR and Westernblot(WB)were used to analyze the changes of proliferation index COLII and differentiation index RUNX2 of chondrocytes after low KANSL1 gene expression.The changes of chondrocyte proliferation and apoptosis were detected by CCK-8 and flow cytometry.Mice were injected with KANSL1 sg RNA lentivirus around the hip to analyze the effect of down-regulated KANSL1 gene expression on hip cartilage development.The changes of hip cartilage were observed by HE and immunofluorescence staining.By detecting the changes of H4K16,H4K5 and H4K8 protein levels in chondrocytes and hip cartilage,we analyzed whether down-regulated expression of KANSL1 gene affected the development of hip cartilage by regulating the epigenetic modification of histidine acetylation.Part Ⅱ: the mechanism of DDH susceptibility caused by KANSL1 gene mutation1.The KANSL1 gene point mutation(P.S265F)detected in DDH family was introduced into m ESCs cells using crisp/cas9 system.After monoclonal culture and identification,the cells were divided into wild group and mutant group.The expressions of Ssea1 and Ssea4 were detected by immunofluorescence to identify embryonic stem cells and the differentiation of their omnipotent.CCK-8 was used to detect changes in the proliferative capacity of the two groups of cells at 7,14 and 21 days.The m RNA expression levels of KANSL1 and MMP13 were detected by Rt-PCR at 7,14 and 21 days.2.C57/B6 mice with KANSL1 gene mutation(p.S256F)were constructed,and the mice were divided into wild group and mutant group after genotype identification.Hip joints of mice were taken 4 weeks and 8 weeks after birth,and the general morphology was observed,and HE staining was performed.The difference of KANSL1,COLII,RUNX2 and MMP13 expression levels in the two groups of mouse cartilage was observed by immunohistochemical staining.Apoptosis level of hip joint chondrocytes was detected by TUNNEL method.Results: Part Ⅰ: The genetic contribution of KANSL1 gene mutation in the pathogenesis of DDH in Han population and the effects of down-regulated KANSL1 gene expression on chondrocyte and hip cartilage development.1.The mutation D744 H in KANSL1 gene of sporadic DDH cases was found to cause damage to protein function.2.The expression levels of proliferation index COLII and differentiation index RUNX2 m RNA in primary chondrocytes after down-regulated KANSL1 gene expression compared with those in the control group.Compared with the control group,the proliferation ability of chondrocytes decreased and the apoptosis level increased.3.After perihip injection of virus to down-regulate KANSL1 gene expression in hip cartilage of mice,hypertrophic cells in cartilage increased and arranged disordered,and COLII expression was down-regulated compared with control group.4.The expression level of H4K16 in primary chondrocytes and mouse hip cartilage with KANSL1 gene down-regulated expression was up-regulated compared with the control group.Part Ⅱ: the mechanism of DDH susceptibility caused by KANSL1 gene mutation1.The expression of Ssea1 and Ssea4,markers representing pluripotent differentiation,did not differ significantly between the wild group and the mutant group after the introduction of KANSL1 gene mutation(P.S256F)into embryonic stem cells.2.m ESCs cells in the mutant group became less in number and irregular in shape after differentiation into chondrocytes.The proliferation rate of cells in the mutant group decreased at 7,14 and 21 days compared with that in the wild group,and there was no significant difference in KANSL1 gene expression compared with that in the wild group,but the expression of MMP13,an indicator of cartilage degeneration,was gradually upregulated over time.3.In the mutant group,the acetabular shape was slightly oval,and the acetabular fossa became shallow.The number of proliferating cells in chondrocyte layer of acetabulum and femoral head in 4-week-old mutant group was lower than that in wild group,and the number of mast cells was increased.At 8 weeks of age,the acetabulum in both groups had only fibrocartilage layer,and the mutant group was slightly thinner than the wild group.Cells in proliferative layer and hypertrophic layer of femoral head were more irregular than those in wild group.4.There was no significant difference in KANSL1 gene expression between wild group and mutant group by immunohistochemical detection.KANSL1 gene expression was found in all layers of cartilage at 4 weeks postnatal,mainly in proliferating cell layer,and rarely expressed in hip cartilage at 8 weeks postnatal.COLII was expressed in all layers of acetabular and femoral head cartilage in both groups at week 4 and 8,and the expression in proliferative and hypertrophic layers in mutant group was significantly reduced compared with that in wild group.RUNX2 was less expressed in the proliferative and hypertrophic layers of acetabular cartilage in the mutant group at week4 than in the wild group,and almost no expression in the acetabular and femoral head cartilage in the two groups at week 8.At the 4th week,MMP13 was mainly distributed in the hypertrophic cell layer and ossification layer in both groups,but it was also distributed in the proliferative layer in the mutant group,which was significantly more than that in the wild group.At 8 weeks,the expression of MMP13 in both groups was significantly increased compared with that at 4 weeks,and MMP13 was more in the mutant group,which was also visible in the resting layer.5.There were few apoptotic chondrocytes in both groups at 4,and 8 weeks,but a small number of apoptotic cells were seen in the hypertropic cell layer and calcification layer in the mutant group.Conclusion:1.KANSL1 gene mutation can lead to the susceptibility of Han population to DDH.2.KANSL1 gene is involved in the development of chondrocytes,and low expression of KANSL1 gene reduces the proliferation and differentiation ability of chondrocytes and acetabular cartilage.KANSL1 gene may affect cartilage development by regulating the epigenetic modification of histidine acetylation.3.The point mutation of KANSL1 gene had no obvious effect on the totipotent differentiation of embryonic stem cells,but affected the number and morphology of chondrocytes.The proliferation ability of cells in the mutant group gradually decreased over time,and showed a trend of degeneration.4.Mutations of KANSL1 gene affect the proliferation and differentiation level of hip cartilage,with accelerated degeneration and slightly increased apoptosis level.