Cardiovascular Phenotype And Its Relationship With Genotypes Of Osteogenesis Imperfecta:clinical And Animal Study

Part 1.Cardiovascular abnormalities and its correlation with genotypes of children with osteogenesis imperfectaBackground and objective:Osteogenesis imperfecta(OI)is an inherited connective tissue disease with increased skeletal fragility,impaired type Ⅰ collagen synthesis and metabolism.Type Ⅰ collagen is an important component of systemic connective tissue and is widely distributed in multiple organs and tissues throughout the body.Therefore,OI patients could also have a plethora of extra-skeletal phenotypes,such as blue sclera,hearing loss,ligament laxity,dentinogensis imperfecta,and valvular heart disease.OI is a complex disease with great heterogeneity in the spectrum of causative genes and clinical phenotypes.Previous studies mostly focused on skeletal phenotypes of OI,but extra-skeletal manifestations could also affect the quality of life in OI patients.Among them,the complication of cardiovascular system could even cause an increased incidence of death in OI patients.Adults with OI have a significantly increased risk of developing cardiovascular diseases.However,previous study about cardiovascular phenotypes in OI patients,particularly in children with OI,were not sufficient,and the relationship between OI genotypes and cardiovascular phenotypes remained unknown.Therefore,the aim of this study is to evaluate the cardiovascular changes in children with OI by echocardiography and electrocardiogram,and to analyze their relationship with genotypes.Methods:A cross-sectional comparative study was completed in a relatively large sample of OI children,who were matched in body surface area(BSA)with healthy controls.All echocardiography was performed by experienced cardiologists using Vivid 7 equipment.The resting standard 12-lead electrocardiogram(ECG)were obtained in OI patients.Clinical phenotypes of OI patients were evaluated,including information of bone fractures,deformities,motility,bone mineral density(BMD),and extra-skeletal phenotypes.Pathogenic mutations of OI were detected by a nextgeneration sequencing(NGS)panel and confirmed by Sanger sequencing.Differences between subgroups of OI(Sillence classification and genotypes of OI)and the control group were evaluated.Then,the respective correlation between OI,OI Sillence types,OI genotypes,and echocardiographic measurements,which adjusted for age,gender,and BSA were analyzed.Results:A total of 69 OI children and 42 healthy children matched in BSA were enrolled.Abnormalities of echocardiography were found in six OI children,including enlarged left atrium(n=5),increased internal diameter of the left ventricle(n=1),who all carried the COL1A1 mutation.Mild regurgitation of mitral or tricuspid valves was observed in 26 OI patients.Abnormal ECG manifestations were found in eight OI children,including deep Q wave,T wave change,premature ventricular complexes,short P-R interval,incomplete bundle branch block and high voltage of left ventricular.Compared with healthy controls,OI children had significant larger values in the main pulmonary artery(MPA)(1.84 vs 1.60 cm,P<0.01),left atrial diameter(2.58 vs 2.11 cm,P<0.001),left ventricular internal dimension at end-diastolic(LVEDd)(3.85 vs 3.50 cm,P<0.05)and lower left ventricular ejection fraction(LVEF)(68.40 vs 71.74%,P<0.01).OI patients with COL1A1 mutation tended to have greater MPA(P<0.01),larger diameters of left atrial(P<0.001)and LVEDd(P<0.05),and lower LVEF(P<0.01)than healthy controls.The alterations of cardiovascular systems were found both in OI children with haplo-insufficiency of type I collagen and with structure change of type I collagen.COL1A1 mutation was independently correlated with dilated MPA(β=1.557,P<0.01),LAD(β=3.915,P<0.001),and LVEDd(β=2.714,P<0.01),and decreased LVEF(β=-3.249,P<0.01).Conclusion:Cardiovascular alterations were identified in OI children,including increased dimensions of the MPA and left chambers,and low LVEF.The cardiovascular abnormalities seemed to be correlated to COL1A1 mutation and defects of type I collagen,which expanded our understandings of the cardiovascular phenotypes of OI children.Part 2:Cardiovascular phenotypes,and its relationship with synthesis and metabolism of collagen in a new rat model of Pls3 related early-onset osteoporosisBackground and objective:A total of 24 causative genes of OI have been identified so far,involving a variety of important signaling pathways of collagen synthesis and metabolism,of which 85-90%of the genes are inherited in a dominant type,and the remaining 10-15%are inherited in a recessive type.Plastin 3(PLS3)is an important protein that regulates the binding and bundling of F-actin,thus involved in a variety of cellular processes,signaling pathways,and diseases.Pathogenic variants in PLS3 have been reported to be associated with early-onset osteoporosis including fractures in men and mild osteoporosis in women,and is considered to be a rare causative gene of OI.However,its relationship with the metabolism of type I collagen remained unknown.There were several cases reported about OI children with PLS3 mutation had patent ductus arteriosus(PDA).Few studies have explored the cardiovascular manifestations of rare PLS3 mutation.In this study,we evaluated the cardiovascular phenotype,collagen synthesis and metabolism in cardiovascular tissues of a novel OI rat model of Pls3 related early-onset osteoporosis.Methods:We used a rat model of large-fragment Pls3 gene knockout(Pls3E1016del/0),which was constructed by our research group using CRISPR/Cas9 technology.Immunohistochemical staining was used to identify the expression pattern of Pls3 in cardiovascular tissues of 6-8 weeks old rat,and the change of Pls3 expression after Pls3 knockout.Pls3E10-16del/0 rats were evaluated for cardiovascular phenotypes,including echocardiography was used to assess their cardiovascular morphology and function,HE stained sections were used to understand their heart and myocardial cells,and immunofluorescence staining of platelet endothelial cell adhesion molecule-1(PECAM-1)was used to evaluate the vascular endothelium.In order to evaluate the expression and structure of collagen in heart and great vessels of Pls3E10-16del/0 rats,Masson staining was used to analyze the expression and distribution of collagen in cardiovascular tissues,and scanning electron microscopy was used to evaluate the collagen microstructure and arrangement.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect whether the expression levels of actin and collagen synthesis-related genes were changed in Pls3E10-16del/0 rats compared with wild-type,in the purpose of exploring the relationship between Pls3 mutation and collagen synthesis and metabolism.Results:Pls3 was widely expressed in the cytoplasm of cardiovascular tissue in wild-type rats,and Pls3 expression in cardiovascular tissue was affected after Pls3 knockdown.In terms of cardiovascular phenotypes,no significant structural and functional abnormalities were observed in the heart and great vessels of Pls3E10-16del/0 rats.However,compared with wild-type rats,Pls3E10-16del/0 rats showed reductions in right ventricular end-diastolic diameter and proximal ascending aorta diameter,and reduced and disorganized vascular endothelial cells.In terms of collagen expression and structure,although the collagen volume fraction in Pls3E10-16del/0 was not significantly changed compared with wild-type rats,scanning electron microscopy revealed that fine collagen fibers were broken and disorganized in the heart and ascending aorta of Pls3E10-16del/0 rats.In addition,the expression levels of actin synthesis gene Acta2,and genes related to collagen synthesis and metabolism,including Col1al,Col3a1.Tgfβ,Smad2 and Smad3,were not significantly changed in Pls3E10-16del/0 rats compared with wild-type rats.Conclusion:In this study,we firstly evaluated the cardiovascular system of a new rat model,Pls3E10-16del/0,which was characterized with early-onset osteoporosis.We found that there were decreased internal diameter of the right ventricle and ascending aorta,reduced vascular endothelial cells,and impairment of collagen fibers in Pls3E1016del/0 rats.However,no significant abnormality of cardiovascular structure and function were found.These findings suggested that Pls3 gene mutation could affect the endothelial cells of vessels,but its relationship with synthesis and metabolism of collagen needed further exploration.This study expanded the understanding of cardiovascular manifestations of OI rats with rare genotypes.