Ⅵ型成骨不全症致病基因突变分析及成骨不全症血清PEDF水平研究

发布时间：2019-01-19 21:35

【摘要】：研究背景：成骨不全症(osteogenesis imperfecta,01)是一种以骨骼脆性增加、骨量减少为特征的单基因遗传病。VI型OI是常染色体隐性遗传、表现为严重骨骼畸形、血清色素上皮衍生因子(pigment epithelium-derived factor, PEDF)含量下降的罕见类型OI。PEDF是一种由SERPINF1基因编码的分泌性糖蛋白,在骨代谢中可能通过多种信号通路调节成骨细胞和破骨细胞活性、并与血管内皮生长因子(vascular epithelial growth factor, VEGF)相互作用从而影响骨骼矿化。研究目的：本研究旨在分析VI型OI患者的表型特征,检测致病基因SERPINF1的突变类型,同时检测134例不同类型OI患者血清PEDF水平,分析其在VI型OI患者诊断和治疗过程中的价值,并探讨PEDF在OI发病机制中可能的作用。研究方法：纳入来自五个非近亲婚配家系的六例OI患者,评估临床表现、血清钙磷及骨转换指标水平和骨骼X线表现。采用双能X线吸收仪(dual energy X-ray absorptiometry, DXA)测量骨密度。采用自主设计的基因芯片,通过二代测序技术进行致病基因突变检测。纳入94例分子诊断明确及41例临床诊断的OI患者、6例SERPINFl基因突变携带者及24例年龄性别匹配的正常对照。采用酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)检测血清PEDF含量。分析血清PEDF水平与患者的年龄、性别、身高、体重、身体质量指数、骨转换指标、25羟维生素D、甲状旁腺激素、骨密度、疾病严重程度及双膦酸盐疗效的相关性。研究结果：六例患者均婴幼儿起病,具有四肢长骨屈曲、脊柱侧凸、后凸等不同程度的骨骼畸形,无蓝色巩膜、牙本质发育不全或听力障碍；影像学表现为严重骨质疏松、长骨干骺端爆米花样改变、骨骼延迟愈合等。致病基因检测提示一例患者为第3外显子纯合突变,c.271 279dupGCCCTCTCG (p.Ala91 Ser93dup);一例患者为第3内含子和第5外显子复合杂合突变,c.283+1GT, c.498_499de1CA (p.Arg167SerfsX35);一例患者为第8外显子纯合突变,c.1202_1203delCA (p.Thr401ArgfsX);一例患者为第3外显子复合杂合突变,c.184GA(p.Gly62Ser), c.271_279dupGCCCTCTCG (p.Ala91_Ser93dup);最后来自同一家系的两例患者中检测到第4外显子杂合突变,c397CT(p.Gln133X)。本研究共检出5种SERPINF1基因的新突变。上述基因突变使成骨细胞分泌的PEDF表达水平显著下降(0.0001-3.93μg/ml)。血清PEDF含量测定提示六例VI型0I患者的血清PEDF含量较年龄、性别匹配的其他基因型和Sillence分型的OI患者、正常对照及SERPINF1基因突变携带者均显著下降(P0.01)；双膦酸盐治疗前后血清P EDF含量、骨转换指标、骨密度无显著变化(P=0.58,0.78,0.11,0.37,0.83)；体重与血清PEDF水平呈正相关(PEDF=6.48+0.378×体重Z值,r2=0.127, P=0.003)。研究结论：本研究首次报道国内SERPINF1基因突变导致罕见的常染色体隐性遗传VI型OI患者的临床特点,在中国患者中检测出五种SERPINF1基因的新型突变。血清PEDF含量显著下降对VI型OI患者具有独特的诊断价值,但PEDF在VI型OI发病机制中的作用有待进一步研究。脂肪细胞能够大量分泌PEDF,故体重可以是血清PEDF水平的影响因素之一。双膦酸盐治疗不影响VI型OI患者的血清PEDF水平、骨转换指标和骨密度,因此新型单克隆抗体类生物制剂在VI型OI的治疗中充满前景。
[Abstract]:Background: The osteogenesis imperfecta, 01 is a single-gene genetic disease characterized by increased bone fragility and osteopenia. Type VI OI is an autosomal recessive inheritance, which is a rare type of OI. PEDF is a secreted glycoprotein encoded by the SERPINF1 gene. Osteoblasts and osteoclast activity may be regulated by a variety of signal pathways in bone metabolism and interact with vascular endothelial growth factor (VEGF) to affect bone mineralization. Objective: To study the phenotypic characteristics of the patients with type VI OI, to detect the mutation type of SERPINF1, and to detect the serum PEDF levels in 134 patients with different types of OI, and to analyze their value in the diagnosis and treatment of type VI OI patients. The possible role of PEDF in the pathogenesis of OI was discussed. Methods: Six patients with OI from the five non-close-of-the-kin-family system were included to assess the clinical manifestations, serum calcium and phosphorus and bone turnover index and bone X-ray performance. Bone mineral density was measured by dual energy X-ray absorption meter (DXA). the gene chip with independent design is adopted, and the pathogenic gene mutation detection is carried out by the second generation sequencing technology. Among the 94 patients with OI, 6 SERPINFl gene mutation carriers and 24 age-matched normal controls were included in 94 cases of molecular diagnosis. The serum PEDF content was detected by enzyme-linked immunosorbent assay (ELISA). The relationship between the serum PEDF level and the patient's age, sex, height, body weight, body mass index, bone turnover index, 25-hydroxyvitamin D, parathyroid hormone, bone mineral density, severity of the disease, and the efficacy of the digluconate were analyzed. The results of the study: 6 patients with infantile onset, with different degrees of skeletal deformity, no blue sclera, dentine hypoplasia, or hearing disorder. The imaging features were severe osteoporosis, and the popcorn-like changes in the long-shaft bone were observed. bone delayed union, and the like. One patient was the third exon-only mutation, c. 271 279dupGCCCTCTCG (p. Ala91 Ser93dup), one case was the third intron and the fifth exon compound hybrid mutation, c. 283 + 1GT, c. 498 _ 499de1CA (p. Arg167SerfX35), and one patient was the eighth exon-only mutation, c. 1202 _ 1203delCA (p. Thr401ArgfsX); One patient was a compound heterozygous mutation in exon 3, c. 184GA (p. Gly62Ser), c. 271 _ 279dupGCCCTCG (p. Ala91 _ Ser93dup), and finally from two patients in the same family to a fourth exon heterozygous mutation, c397CT (p. Gln133X). A new mutation of five SERPINF1 genes was detected in this study. The above-mentioned gene mutation significantly decreased the level of PEDF expression in osteoblasts (0.0001-3.93. mu.g/ ml). The serum PEDF content of 6 patients with type VI 0I was determined by the content of PEDF, and the serum PEDF content in the patients with type VI type I was significantly lower than that of the other genotypes (P0.01). The serum P-EDF content and bone turnover index before and after the treatment of the bivalate were significantly decreased (P0.01). There was no significant change in bone mineral density (P = 0.58, 0.78, 0.11, 0.37, 0.83), and the body weight was positively correlated with the serum PEDF level (PEDF = 6.48 + 0.378, body weight Z, r2 = 0.127, P = 0.003). Conclusion: This study first reported the clinical characteristics of the rare autosomal recessive VI-type OI patients with SERPINF1 gene mutation in China, and the new mutation of five SERPINF1 genes was detected in Chinese patients. The significant decrease in serum PEDF content has a unique diagnostic value for patients with type VI OI, but the role of PEDF in the pathogenesis of type VI OI is to be further studied. The fat cells can secrete PEDF in a large amount, so the body weight can be one of the factors of the serum PEDF level. The treatment of bivalate does not affect the serum PEDF level, bone turnover index and bone mineral density of the patients with type VI OI, so the novel monoclonal antibody-based biological preparation is full of prospect in the treatment of the VI-type OI.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R681.1

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