Pathological Characteristics And Autosomal Recessive Mutations Of Chinese Osteogenesis Imperfecta Patients

Background and Objectives:Osteogenesis imperfecta （OI）, also known as brittle bone disease, is mainly characterizedby bone fragility, frequent fractures and reduced bone mass. Blue sclera, dentinogenesisimperfecta, loose skin, muscle weakness, joint laxities are frequently observed in OI patients.Approximately,90%of OI patients are caused by mutations of COL1A1or COL1A2genes.Mutations in these genes lead to the abnormal structure or low percentage of type I collagene.In the histopathologic features aspect, OI patients usually characterized by decreased dermalcollagen, muscle fiber atrophy, sparse and thinner trabecular with low density. Currently,histopathology study concerning OI is very limited for its rare prevalence as well asunavailable tissue samples. By collaboration with Chinese excellent clinical OI experts, wecollected skin, skeletal muscle and bone tissues form OI patients receiving clinical orthopedicoperation. Quantifying and qualitative study of its histopathological features is conductedunder the obtained informed consent and the approval from the Ethical Committee ofShandong Academy of Medical Sciences in this study.Methods：Fifty skin, skeletal muscle, and bone tissue specimens of dislocation of the hip （DDH）patients collected during surgical operation were selected as control group and74OI patientswere selected as the experimental group. All children patients recruited aged0-6years old.HE and Weigert-Van Gieson staining were conducted to display the pathological elastic fibersand collagen fibers of skin. HE and Masson trichromatic staining was performed to distinguishthe collagen fibers and muscle fibers from63cases of skeletal muscle tissue and107cases ofbone tissue. Further quantitative analysis was followed by measuring epidermis and dermis thickness, epidermis cell layers thickness, papillary and reticular layer dermis collagen fiberdiameter, dermal collagen and elastic fiber ratio;muscle density and diameter; trabecularthickness, bone lacuna diameter and density, area ratio of cancellous bone marrow cavity,cortical bone osteon and the central tube diameter. from three tissues mentioned above usingImage-Pro Plus6.0software. Statistical analysis of data was completed by SPSS17.0. Inaddition, we observed the ultrastructure of bone tissue from one OI type V patient bytransmission electron microscopy.Results：Compared with the DDH control group, reduced skin thickness was observed in15of74OI patients, sparse collagen fibers of reticular dermis in15cases, increased fat tissue in3cases,hyperplasia and fragments of elastic fibers in8patients. The remaining samples showed noobvious abnormal features. Quantitative analysis demonstrated significantly decreased dermalthickness, diameter of the reticular dermis collagen, at the rates of82.62%,80.40%and87.74%in comparision with DDH control, respectively. While there’s no significantdifference observed in the thickness of the full epidermis layer, epidermal cell layer andpapillary dermis collagen diameter observed in OI and DDH patients. Their values were92.33%,94.57%and97.85%as the DDH control, respectively. The percentage ratio of elasticfiber was3%-8%in DDH group and8%-11%in8OI patients, though there’s no significantdifference noticed.Totally,22of63OI patients showed varying degrees of muscle fiber atrophy, partialmuscle fat hyperplasia, dissolved sarcoplasmic, thinner and uneven thickness of muscle fibers.To quantifying density and diameter of muscle fiber, patients were divided into four agegroups. The results showed the reduced values of density and diameter for muscle fiber. ForOI patients group aged0-1.5and3.1-4.5years old, the values of muscle fiber density was87.65%and85.71%as the age matched DDH control groups. The diameter of muscle fiberfor OI patients aged4.6-6.0years old was82.17%as the control group, these abovedifferences were statistically significant. Values of density and diameter for muscle fiber inother OI groups were slightly decreased but no statistically differences were observed.Under the observation of light microscope,79.44%bone tissue derived from OI patientsappeared or partly appeared varying degrees of abnormalities, which including irregularcortical bone lamellar structure, disorganized and decreased bone collagen fiber, abnormal mineralization of bone matrix, irregular, thinner and sparse trabecular, incomplete osteon,more enlarged lacunae and increasement in porosity, medullary cavity, woven bone and thenumber of osteoblasts.In comparision with DDH control group, quantitative analysis showed that the trabecularthickness decreased significantly about29.66%. Bone lacuna diameter and area ratio ofcancellous bone marrow cavity significantly enlarged with the percentage of43.57%and23.46%respectively. In addition, the diameter of cortical bone osteon and the central tubedecreased12.29%and13.30%, respectively, while the density of bone lacunas increased4.64%. Though there’s no statistically significant differences observed in all three valuesabove. Electron microscopy observation showed irregular collagen fibers, much bone tubesand proteoglycan surrounding the osteocytes in bone specimen from one OI type V patient.Conclusion:The pathological changes of bone tissue are extremely obvious and more prevalent thanchages in skeletal muscle and skin specimens. Quantitative analysis discovered that someparameters related with the skin, skeletal muscle and bone tissues can be taken as thepathological diagnosis index of OI disease. Though the study based on the large samples,further study, like the relationship between the parameters and disease severity, OI subtypesare still needed. Background and objectives:Osteogenesis imperfecta （OI） is a group of connective tissue diseases with bothphenotypic and genetic heterogeneity. Till now,15OI subtypes have been identified. TypeI-IV is classified based on the clinical severity as well as imaging changes. Type V-XV isnominated according to the pathogenic genes. Autosomal dominant OI is the main inheritanceform and COL1A1, COL1A2genes that encode the pro-α chains are responsible for OIpathogenicity. Less than10%of OI patients is inherited in autosomal recessive pattern.Though rare, twelven different genes have been reported to be related with this autosomalrecessive inherited OI. Generally speaking, OI patient with autosomal recessive inheritanceare more clinical severe. The molecular meachnisms for these autosomal recessive OI genesare complex and they are involved in the folding, assembly, secretion and other pathwaysduring the process of collagen synthesis and metabolism.Originally, our laboratory identified31OI patients without gene mutations for COL1A1,COL1A2and IFITM5autosomal domiant genes based on traditional Sanger sequencing usingperipheral blood DNA samples. The study aims to further detect the left twelven autosomalrecessive OI genes, including SERPINF1, LEPRE1, PPIB, SERPINH1, FKBP10, OSX, BMP1,TMEM38B, WNT1, PLOD2, CREB3L1and CRTAP in these non-classic OI patients.Methods:Peripheral blood genomic DNA was extracted from both patients and their familymembers. All exons of12known autosmal recessive OI genes were amplified by PCRsystems that were optimized in our laboratory’s previous work. PCR products were subjectedto Sanger sequencing. All genetic variations were analyzed by Mutation Surveyor4.0.6software and further confirmed in both the human collagen mutation database and human SNP database. In silico predication of potential mutation effects on protein functions wasperformed using PolyPhen, Align GVGD, SIFT and Mutation Taster online softwares.Mutations were localized in their corresponding three-D structures by Swiss PDB ViewerV4.1. Meanwhile, clinical phenotypes of OI patients with identified gene mutations weredescribed in the study.Results:Among all31OI patients, six of them were found to have WNT1mutations and2haveSERPINF1mutations. There were no pathogenic mutations were found in all remaining23cases of OI patients. In all six WNT1mutations, four of them were compound heterozygousmutations, including c.301C>T+c.681C>A （p.Arg101Cys+p.Cys227X）, c.681C>A+c.1010G>C （p.Cys227X+p.Arg337Pro） and c.397G>A+c.677C>T （p.Ala133Thr+p.Ser226Leu）（twopatients from the same family）. Two homozygous mutations were c.677C>T（p.Ser226Leu）and c.466delC （p.Arg156Glyfs*43）. For type VI OI patients with SERPINF1mutations,c.245₂46het_delAG+c.397C>T（p.Leu83Glnfs*28+p.Gln133X） heterozygous mutation andc.907C>T （p.Arg303X）homozygous mutation were detected. Sydromes of patients mainlyinvolved with long bone and spine, in silico predication indicated the mutations could affectnormal function and lead to the3D structure functional changes of encoding protein.Conclusions:Type XV OI patients with WNT1and VI OI patients with SERPINF1gene mutations areprevalent in autosomal accessive Chinese OI patients. The left samples failed to findmutations for all known OI genes laid the foundation for identification of novel OI candidategenes based on next-generation sequencing technique.