第一部分 成骨不全症基因型-表型相关性及东西方队列特征比较研究

【研究背景和目的】

成骨不全症（Osteogenesis imperfecta, OI）是一种以骨密度降低、骨骼脆性增加和反复骨折为特征的遗传性骨病。OI是最常见的单基因遗传性骨病，在新生儿中的发病率约为1/15000~1/20000。根据国家统计局发布的2022和2023年新生儿出生率数据，预估我国每年出生的OI患儿约有450~700例。由此可见，我国每年新增OI患儿并不少见。然而目前对大样本OI患者骨骼表型的深入研究较少，同时，东西方OI队列是否存在差异也尚不清楚。本部分研究的目的是探究大样本OI队列的基因型和表型及其相关性，并比较东西方国家不同OI患者队列的特征差异。

【研究方法】

纳入2007年1月到2022年5月就诊于北京协和医院内分泌科的671例OI患者（儿童：457例，成人：214例）。采用二代靶向测序检测OI致病性突变，并通过Sanger测序验证。收集患者临床表型信息，采用双能X线骨吸收仪检测骨密度，行X线摄片评估骨折和骨骼畸形情况，并进行基因型-表型相关性分析。检索西方国家OI队列研究，比较东西方OI特征的差异。

【研究结果】

本部分研究在671例临床诊断为OI的患者中发现560例携带OI致病突变，阳性检出率为83.5%。本部分研究共检测出15种OI候选基因，其中COL1A1（n=308, 55%）和COL1A2 （n=164, 29%）基因突变最为常见，SERPINF1和WNT1是最常见的双等位突变基因。此外，我们分别在10个和6个不相关家系中，发现COL1A1 c.769G>A（p.G257R）和COL1A2 c.1009G>A（p.G337S）突变最为常见，提示这两个位点可能是国人OI的热点突变。在414个先证者中，I型OI占48.8%，Ⅲ型占16.9%，Ⅳ型占29.2%，Ⅴ型占5.1%。外周骨折是最常见的临床表型，占96.6%，以股骨骨折最为常见（34.7%）。43.5%的OI患者有椎体压缩性骨折。基因型-表型相关性分析显示：双等位基因突变或COL1A2突变患者比COL1A1突变患者的骨骼畸形和活动困难更严重（P<0.05）。Ⅰ型胶原甘氨酸替代突变或双等位基因突变患者临床表型比Ⅰ型胶原数量减少患者更为严重。Ⅰ型胶原甘氨酸替代突变和双等位基因突变患者的表型相似。此外，Ⅰ型胶原甘氨酸突变位置与Sillence分型呈显著正相关（r = 0.184，P = 0.032），与LS BMD Z值存在负相关趋势（r = -0.158，P=0.073）。同时，我们发现不同国家的OI基因突变谱不同，但东西方OI患者队列的骨折发生率相似。

【研究结论】

本部分研究绘制了大样本OI患者队列的致病基因谱和表型谱，阐述了基因型-表型的相关性，并比较了东西方患者表型和基因型的异同，研究结果对OI的精准诊疗、机制探索和预后判断具有重要价值。另外，不同种族的OI患者遗传特征存在差异，但其机制需要进一步探索。

第二部分 地舒单抗与唑来膦酸治疗成人成骨不全症的疗效及其与基因型和表型相关性研究

【研究背景和目的】

成骨不全症（Osteogenesis imperfecta, OI）是最常见的单基因遗传性骨病，具有明显表型和基因型异质性。目前已知OI致病基因至少26种，绝大多数是由编码Ⅰ型胶原的COL1A1和COL1A2基因突变所致。由于骨的机械强度受损，OI患者各个年龄段骨折风险均高于正常同龄人。OI骨折发生风险在儿童期最高，成年后逐渐下降，因此临床上往往忽视对于成人OI患者的治疗。双膦酸盐是有效的骨吸收抑制剂，目前已被广泛用于儿童OI的治疗。然而，双膦酸盐对严重OI患者疗效较差，因此，迫切需要探索新的OI治疗药物。地舒单抗是一种靶向核因子κ B受体活化因子配体的单克隆抗体，广泛用于治疗绝经后骨质疏松症和男性骨质疏松症。然而，地舒单抗对成人OI患者的疗效和安全性尚不清楚。因此，本部分研究的目的是探索并比较地舒单抗与唑来膦酸对成人OI患者的疗效和安全性, 并评估不同基因型和临床表型对地舒单抗和唑来膦酸的治疗反应。

【研究方法】

这是一项为期12月、前瞻性、开放标签研究。纳入的OI患者随机接受皮下注射60mg 地舒单抗，每6月一次，或静脉输注5mg唑来膦酸一次。采用二代靶向测序检测OI致病性突变，并通过Sanger测序验证。在基线、6和12月时，采用双能X线骨吸收仪检测面积骨密度（Areal bone mineral density, aBMD）和骨小梁评分（Trabecular bone score, TBS），并检测骨转换生物标志物（Bone turnover biomarkers, BTMs）包括β-1型胶原羧基末端肽（β-isomerized carboxy-telopeptide of type I collagen, β-CTX）和碱性磷酸酶（Alkaline phosphatase, ALP）。评估aBMD、TBS和BTMs较基线的变化。同时评估治疗安全性指标和基因型/Sillence分型-治疗反应相关性。

【研究结果】

本部分研究共纳入51名OI成人患者（地舒单抗组：25名，唑来膦酸组：26名），基因检测发现其中49名患者存在OI致病性突变（地舒单抗组：25例，唑来膦酸组：24例）。两组患者的基线临床特征一致。治疗12月，地舒单抗组腰椎和全髋的aBMD分别增加了4.34%（P=0.005）和1.45%（P=0.023），而唑来膦酸组分别增加了4.92%（P=0.006）和2.02%（P=0.016）。同时，治疗12月，两组TBS分别增加了1.39%和2.70%，然而未达到统计学意义。地舒单抗治疗6和12月，血清骨吸收指标β-CTX水平分别下降49.71 ± 9.20%（P=0.002）和45.64 ± 10.45%（P=0.010）；血清骨形成指标ALP 水平分别下降了21.61% ± 19.23%（P=0.001）和22.02 ± 20.58%（P=0.004）。唑来膦酸治疗6和12月，血清β-CTX水平分别下降59.88 ± 24.61%（P=0.010）和49.18 ± 35.51%（P=0.025），血清ALP水平分别下降了29.80 ± 14.41%（P=0.012）和26.34 ± 22.44%（P=0.053）。两组在治疗期间aBMD、TBS和BTMs的变化百分比无统计学差异。进一步的基因型-治疗反应分析显示：地舒单抗治疗12月，轻型OI患者TBS增长率显著高于重型OI患者（P=0.030）。在研究期间，地舒单抗组的不良反应发生率低于唑来膦酸组。

【研究结论】

地舒单抗可有效抑制成人OI患者骨吸收，增加aBMD，其疗效与唑来膦酸相似。未来需要开展长期和大样本的研究来证实地舒单抗在成人OI患者中的抗骨折疗效和安全性。

第三部分 骨质疏松症患者的蛋白质组学研究

【研究背景和目的】

骨质疏松症是一种以低骨量和骨组织微结构破坏为特征的代谢性骨病，最严重的后果是导致轻微外力下骨折。骨质疏松症发病的基础是骨重塑的失衡，然而目前对骨重塑失衡导致骨质疏松症的分子机制仍然知之甚少。尽管既往研究通过蛋白质组学技术发现了部分调控骨代谢的因子，但研究结果存在不一致，同时，性别差异对骨质疏松症发病机制和蛋白表达变化是否有影响也尚不清楚。因此，本部分研究的目的是利用骨组织蛋白质组学技术，分别探索女性和男性骨质疏松症发病相关的蛋白表达变化和可能的调控信号通路，并对差异蛋白进行验证，明确骨质疏松症发病的可能调控因子，以指导进一步的机制探索。

【研究方法】

本部分研究是一项横断面研究。纳入于北京协和医院手术的骨质疏松性骨折和性别匹配的非骨质疏松性骨折患者，收集患者病史资料，检测骨密度（Bone mineral density，BMD）、骨转换指标（Bone turnover biomarkers，BTMs）和常规生化指标。获取患者骨骼标本，采用基于液相色谱-串联质谱的非靶向蛋白质组学技术对女性和男性骨骼标本进行分析并筛选差异表达蛋白。对筛选的差异蛋白进行生物信息学分析，探索分子通路，寻找核心差异表达蛋白，通过受试者工作特征（Receiver operator characteristic，ROC）曲线评估核心差异蛋白与骨质疏松症的相关性强度，并分析核心差异蛋白与BMD和BTMs的相关性。进一步收集其他符合纳入标准的女性和男性患者的骨组织标本，通过Western blot和免疫组化验证核心差异蛋白表达。

【研究结果】

本部分研究纳入女性骨质疏松性骨折患者（病例组）13例，非骨质疏松性骨折患者（对照组）8例；男性骨质疏松性骨折患者（病例组）12例，非骨质疏松性骨折患者（对照组）7例。女性和男性病例组年龄均高于对照组（P<0.001），腰椎、股骨颈和全髋BMD均低于对照组（P<0.01）。通过数据非依赖性采集模式检测蛋白，以差异倍数>2.0或<0.5和P值< 0.05两个条件作为显著性差异蛋白的筛选标准。在女性病例-对照比较组中共鉴定到1161种差异表达蛋白，其中病例组上调蛋白917种，下调蛋白244种；男性病例-对照比较组共鉴定到786种差异表达蛋白，其中病例组上调蛋白560种，下调蛋白226种。

根据每组差异蛋白的表达情况，删除缺失值≥50%的蛋白。对女性Top 245和男性Top 155种上调蛋白进一步分析，结果显示女性和男性蛋白质组呈现出不同的特征，涉及不同的生物学功能和通路。女性骨质疏松性骨折主要富集于吞噬体、溶酶体、整合素介导的细胞黏附、抗原加工和提呈及代谢途径等通路，提示女性骨质疏松症主要与“免疫炎症反应”相关；男性骨质疏松性骨折主要富集于氧化磷酸化、活性氧、线粒体呼吸链复合体I的组装及线粒体电子传递及细胞呼吸等通路，这些通路主要与“氧化应激反应”相关。

进一步分析提示女性7种核心上调蛋白（HLA-A、ITGB2、HLA-DRB1、ATP6V0A1、HLA-C、HLA-DPB1和HLA-DRA）以及男性10种核心上调蛋白（NDUFA11、COX5B、NDUFA8、COX7A2、NDUFAB1、NDUFA3、UQCRC1、COX5A、NDUFV2和VDAC1）可能参与骨质疏松症的发病。ROC曲线分析发现这些核心上调蛋白与骨质疏松存在较强关联（女性：AUC为0.903，95% CI：0.718~1；男性：AUC为0.851，95% CI：0.366~1），相关性分析显示核心上调蛋白与BMD呈显著负相关（P<0.05），而与P1NP或β-CTX呈显著正相关（P<0.05）。

采用Western blot和免疫组化验证核心差异蛋白，结果显示HLA-C在女性病例组表达显著高于对照组（P=0.035）。ITGB2在女性病例组表达趋势高于对照组，而COX7A2在男性病例组表达趋势也高于对照组，但两者均未达到统计学差异，未来仍需扩大样本量进一步验证。

【研究结论】

本部分研究揭示了女性和男性骨质疏松症发病的可能分子通路，发现女性和男性骨质疏松症的差异蛋白表达谱及其生物学功能存在不同，本部分研究有助于理解骨质疏松症发病机制的性别差异，也为发现新的治疗靶点和制定个性化精准治疗策略提供基础。未来需要更广泛的样本和深入的功能性研究进一步验证。

Part 1 Genotype–phenotype relationship and comparison between eastern and western patients with osteogenesis imperfecta

【Background and Purpose】

Osteogenesis imperfecta (OI) is an inherited bone disease characterized by decreased bone density (BMD), increased bone fragility, and recurrent fractures. OI is the most common monogenic hereditary bone disorder, with an incidence of approximately one in 15 000~20 000 births. Based on the birth rate data for 2022 and 2023 released by the National Bureau of Statistics, it is estimated that approximately 450~700 Chinese children are born with OI, indicating that the annual addition of new OI children in China is not uncommon. However, an in-depth study regarding genotypic and skeletal phenotypic relationships in a large cohort of Asian OI patients has not yet been reported. Additionally, it remains unclear whether differences in phenotypes and genotypes exist between Eastern and Western OI patients. Therefore, we aimed to investigate the genotypes and phenotypes of a large cohort of OI, to explore their relationship, and to compare the differences between Eastern and Western OI cohorts.

【Methods】

A total of 671 OI patients were included from January 2007 to May 2022 (children: 457; adults: 214). Pathogenic mutations were detected using next-generation targeted sequencing and verified through Sanger sequencing. Phenotypic information was collected, and BMD was measured using dual-energy X-ray absorptiometry, fractures and skeletal deformities were assessed by radiography, and genotype-phenotype correlations were analyzed. Literature about Western OI cohorts was searched, and differences were compared between Eastern and Western OI cohorts.

【Results】

Among 671 patients clinically diagnosed with OI, 560 were found to carry OI-causing mutations, with a positive detection rate of 83.5%. A total of 15 candidate genes for OI were identified, among which mutations in COL1A1 (n=308, 55%) and COL1A2 (n=164, 29%) were the most common, with SERPINF1 and WNT1 being the most prevalent biallelic mutant genes. Additionally, mutations in COL1A1 c.769G>A (p.G257R) and COL1A2 c.1009G>A (p.G337S) were discovered in 10 and 6 unrelated families, respectively, which were the most common mutation sites, suggesting these mutations may be hotspots for OI in the Chinese population. Among 414 probands, type I OI accounted for 48.8%, type Ⅲ for 16.9%, type Ⅳ for 29.2%, and type Ⅴ for 5.1%. Peripheral fractures were the most common clinical phenotype, occurring in 96.6% of OI patients, with femoral fractures being the most common (34.7%). Vertebral compression fractures were present in 43.5% of OI patients. Genotype-phenotype correlation analysis showed that patients with biallelic variants or COL1A2 mutations had more severe skeletal deformities and mobility difficulties compared to those with COL1A1 mutations (all P<0.05). Glycine substitution of COL1A1 or COL1A2 or biallelic variants led to more severe phenotypes than haploinsufficiency of collagen type I α chains, which induced the mildest phenotypes. Further, the phenotypes of type I collagen glycine substitution mutations and biallelic mutations were similar. In addition, the position of glycine mutation in type I collagen showed a significantly positive correlation with Sillence type (r = 0.184, P= 0.032), and negative correlation trend with LS BMD Z value (r = -0.158, P=0.073). Furthermore, it was found that although the gene mutation spectrum varied among countries, the fracture incidence was similar between Eastern and Western OI cohorts.

【Conclusion】

This study delineates the genotypic and phenotypic spectrums in a large cohort of OI patients, elucidates the correlations between genotype and phenotype, and compares the similarities and differences in phenotypes and genotypes between Eastern and Western OI cohorts. These findings are significant for the precision diagnosis and treatment of OI, the exploration of its mechanisms, and the prognosis determination. Additionally, the genetic profiles of OI patients may vary among races, but the mechanism needs to be explored.

Part 2 Efficacy of denosumab and zoledronic acid on adult patients with osteogenesis imperfecta and their relationship with genotype and phenotype

【Background and Purpose】

Osteogenesis imperfecta (OI) is the most common monogenic inherited skeletal dysplasia that is phenotypically and genetically heterogeneous. Currently, at least 26 causative genes for OI have been identified, with the majority resulting from mutations in the COL1A1 and COL1A2 genes. Due to compromised mechanical strength of bones, individuals with OI have a higher risk of fractures compared to the healthy population. The fracture incidence is highest during childhood and then declines in midlife. Therefore, the treatment of OI adults is often neglected in clinical practice. Bisphosphonates, potent inhibitors of bone resorption, are currently widely used in the treatment of pediatric OI. However, their efficacy in patients with severe OI is relatively poor. Thus, there is an urgent need to explore new pharmacological treatments for OI. Denosumab, a monoclonal antibody targeting receptor activator of nuclear factor-κ B ligand, is extensively used for the treatment of postmenopausal osteoporosis and male osteoporosis. However, the efficacy and safety of denosumab in adult patients with OI remain unclear. Therefore, the aim of this study was to investigate and compare the efficacy and safety of denosumab versus zoledronic acid in adult patients with OI, and to evaluate the treatment response of different genotypes and clinical phenotypes to denosumab and zoledronic acid.

【Methods】

This was a 12-month, prospective, randomized, open-label study. Adult patients with OI were randomly assigned to receive either a subcutaneous injection of 60 mg denosumab every 6 months or a single intravenous infusion of 5 mg zoledronic acid. Pathogenic mutations of OI were identified using next-generation targeted sequencing and validated via Sanger sequencing. Areal bone mineral density (aBMD) and trabecular bone score (TBS) were measured using dual-energy X-ray absorptiometry at baseline, 6 months, and 12 months, and the changes from baseline of aBMD and TBS were evaluated. Additionally, the percentage changes in bone turnover biomarkers (BTMs) including β-isomerized carboxy-telopeptide of type I collagen (β-CTX) and alkaline phosphatase (ALP), as well as safety parameters and genotype/Sillence type- treatment response associations were assessed.

【Results】

A total of 51 OI adults (denosumab: 25, zoledronic acid: 26) were included, of whom 49 patients had identified pathogenic mutations (denosumab: 25, zoledronic acid: 24). Baseline clinical characteristics were consistent between both groups. After 12 months of treatment, the denosumab group exhibited increases in aBMD at the lumbar spine and total hip by 4.34% (P=0.005) and 1.45% (P=0.023), respectively, while the zoledronic acid group showed increases of 4.92% (P=0.006) and 2.02% (P=0.016), respectively. TBS showed an increasing trend by 1.39% and 2.70% in denosumab and zoledronic acid groups, respectively.

Following 6 and 12 months of denosumab treatment, levels of the bone resorption marker β-CTX decreased by 49.71 ± 9.20% (P=0.002) and 45.64 ± 10.45% (P=0.010), respectively. Levels of the bone formation marker ALP decreased by 21.61% ± 19.23% (P=0.001) and 22.02 ± 20.58% (P=0.004), respectively. In the zoledronic acid group, serum β-CTX levels decreased by 59.88 ± 24.61% (P=0.010) and 49.18 ± 35.51% (P=0.025) after 6 and 12 months, respectively, while serum ALP levels decreased by 29.80 ± 14.41% (P=0.012) and 26.34 ± 22.44% (P=0.053), respectively. No statistically significant differences were observed in the percentage changes in aBMD, TBS, and BTMs between the two groups during treatment period. Further analysis revealed that, after 12 months of denosumab treatment, OI patients with milder phenotypes showed a significantly higher increase in the TBS after 12 months of denosumab treatment than those with more severe phenotypes (P=0.030). During the study period, denosumab group had fewer adverse events than zoledronic acid group.

【Conclusion】

Denosumab effectively inhibits bone resorption and increases aBMD in OI adults, with similar efficacy to zoledronic acid. Long-term and large-sample studies are needed to confirm the anti-fracture efficacy and safety of denosumab in adult OI patients.

Part 3 Proteomic study of patients with osteoporosis

【Background and Purpose】

Osteoporosis is a metabolic bone disease characterized by low bone mass and deteriorated bone microarchitecture, leading to fractures under minor trauma. The pathological basis for osteoporosis is the imbalance of bone remodeling. However, the molecular mechanism of bone remodeling imbalance leading to osteoporosis is still poorly understood. Although previous studies have found some factors regulating bone metabolism through proteomics technology, the results were inconsistent. Additionally, the impact of gender differences on the pathogenesis and protein expression change of osteoporosis is still unclear. Therefore, the objective of this study was to utilize proteomics to explore proteins expression changes and potential regulatory signaling pathways associated with osteoporosis in both females and males, and to verify the differential proteins, so as to clarify the possible regulators of osteoporosis and guide further mechanism exploration.

【Methods】

This was a cross-sectional study. Patients with osteoporotic fractures and gender-matched non-osteoporotic fractures underwent surgery in Peking Union Medical College Hospital were included. Medical history data of patients were collected. Bone mineral density (BMD), bone turnover biomarkers (BTMs) and routine biochemical indicators were detected. Bone specimens from the patients were obtained, and untargeted proteomics based on liquid chromatography tandem-mass spectrum were used to analyze female and male bone specimens and screen for differentially expressed proteins. Bioinformatics analysis of the selected differential proteins was performed to explore the molecular pathways and find core differential expression proteins. The correlation strength between core differential proteins and osteoporosis was evaluated by Receiver operator characteristic (ROC) curve, and the correlation between core differential proteins with BMD and BTMs was analyzed. Additional bone tissue of female and male patients meeting the inclusion criteria were further collected for validation of the core differential proteins through western blot and immunohistochemistry.

【Results】

This study included 13 female patients with osteoporotic fractures (case group) and 8 female patients with non-osteoporotic fractures (control group); 12 male patients with osteoporotic fractures (case group) and 7 male patients with non-osteoporotic fractures (control group). The ages of both female and male case groups were higher than those of the control groups (P<0.001), and the BMDs at the lumbar spine, femoral neck, and total hip were lower than those of control group (P<0.01). Proteins were detected using the data independent acquisition mode, and significantly different proteins were screened based on a fold change (Fc) > 2.0 or < 0.5 and a P value < 0.05. A total of 1161 differentially expressed proteins were identified in the female case-control group, of which 917 proteins were up-regulated and 244 proteins were down-regulated; 786 differentially expressed proteins were identified in the male case-control group, of which 560 proteins were up-regulated and 226 proteins were down-regulated in case group.

Proteins with missing values ≥50% were excluded based on the expression of differential proteins of each group. Further analysis of the top 245 up-regulated proteins in females and top 155 in males revealed different proteomic characteristics between genders, involving different biological functions and pathways. Female osteoporotic fractures were mainly enriched in pathways such as phagosome, lysosome, integrin-mediated cell adhesion, antigen processing and presentation, and metabolism pathways, suggesting that "immune-inflammatory response" is primarily associated with osteoporosis in females. Male osteoporotic fractures were mainly enriched in pathways related to oxidative phosphorylation, reactive oxygen species, assembly of mitochondrial respiratory chain complex I, and mitochondrial electron transport and cellular respiration, which are mainly associated with "oxidative stress response".

Further analysis suggested that 7 core up-regulated proteins in females (HLA-A, ITGB2, HLA-DRB1, ATP6V0A1, HLA-C, HLA-DPB1 and HLA-DRA) and 10 core up-regulated proteins in males (NDUFA11, COX5B, NDUFA8, COX7A2, NDUFAB1, NDUFA3, UQCRC1, COX5A, NDUFV2 and VDAC1) may be involved in the pathogenesis of osteoporosis. ROC curve analysis found these core up-regulated proteins were strongly associated with osteoporosis (females: AUC = 0.903, 95% CI: 0.718–1; males: AUC = 0.851, 95% CI: 0.366–1). Correlation analysis showed that core up-regulated proteins were significantly negatively correlated with BMD (P<0.05), and significantly positively correlated with P1NP or β-CTX (P<0.05).

Western blot and immunohistochemistry were used to validate the differential proteins. The results showed that the expression of HLA-C in the female case group was significantly higher than that in the control group (P=0.035); ITGB2 exhibited a trend of higher expression in the female case group compared to the control group, while COX7A2 showed a similar trend of higher expression in the male case group compared to the control group, although these differences did not reach statistical significance. Further validation with an expanded sample size is needed in the future.

【Conclusion】

This study revealed potential molecular pathway of osteoporosis in both females and males, identifying distinct proteomic expression profiles and biological functions associated with osteoporosis between the genders. These findings help to understand the gender differences in the pathogenesis of osteoporosis and provides a basis for discovering new therapeutic targets and developing personalized precision treatment strategies. Future studies will require broader sample sizes and more in-depth functional research for validation.