【摘要】：第一部分中國(guó)大樣本成骨不全癥的基因突變譜探索目的:成骨不全癥(Osteogenesis Imperfecta,OI)是一組有高度遺傳和表型異質(zhì)性的單基因遺傳性骨病,以骨量減少、骨脆性增加和反復(fù)骨折為主要特點(diǎn)。目前我國(guó)O1致病基因和潛在分子機(jī)制尚不清楚,相關(guān)臨床表型研究也較為缺乏。本研究采用新型分子診斷平臺(tái),深入探索中國(guó)大樣本OI患者的致病基因突變譜,并分析多種遺傳模式O1患者的基因型與臨床表型關(guān)系。方法:本研究利用自主設(shè)計(jì)、包含19種OI致病基因的二代靶向測(cè)序平臺(tái),對(duì)2013-2016年在北京協(xié)和醫(yī)院就診的152例OI患者進(jìn)行致病基因突變檢測(cè),采用Sanger測(cè)序?qū)γ坷颊哌M(jìn)行家系驗(yàn)證。此外,納入本課題組既往行基因檢測(cè)的O1患者,在共261例OI患者中總結(jié)全面的致病基因突變譜;分析不同基因型患者,身高、骨折率、腰椎和股骨頸骨密度(bone mineral density,BMD)、骨轉(zhuǎn)換生化指標(biāo)血堿性磷酸酶(alkaline phosphatase,ALP)和β膠原降解產(chǎn)物(cross-linked C-telopeptide of type I collagen,β-CTX)、骨骼畸形、骨骼外表現(xiàn)等表型差異;對(duì)常見的Ⅰ型膠原基因變異患者,進(jìn)行深入的基因型-表型關(guān)聯(lián)分析。結(jié)果:本研究利用二代測(cè)序聯(lián)合家系Sanger測(cè)序,發(fā)現(xiàn)了 140例OI患者的致病基因突變,并檢出50種新突變位點(diǎn);新型分子診斷平臺(tái)對(duì)OI的檢出率為92%,準(zhǔn)確性為100%。在本課題組261例OI患者中,Ⅰ型膠原編碼基因(COLlA1/COLlA2)突變最為常見(198例,76%),其次是IFITM5基因(15例,6%),SERPINF 和WNT1(12例,4.6%)是常染色體隱性遺傳性O(shè)I中檢出率較高的兩種致病基因。然而在輕型OI中,Ⅰ型膠原基因檢出率為84%;在中重型OI中,Ⅰ型膠原基因檢出率僅68%,但隱性遺傳基因可達(dá)21%。相比于Ⅰ型膠原結(jié)構(gòu)變異,COL1A/單倍劑量不足患者身高Z值(-0.4±1.3)和BMDZ值(腰椎-1.7±1.3、股骨頸-2.7±2.2)更高,骨骼畸形更少(11%,P均0.05)。Ⅰ型膠原αl鏈結(jié)構(gòu)變異位點(diǎn)與藍(lán)鞏膜、牙本質(zhì)發(fā)育不全,α2鏈變異位點(diǎn)與股骨頸BMD Z值存在一定相關(guān)性;但未發(fā)現(xiàn)氨基酸替換種類與表型的相關(guān)性。所有常染色體隱性遺傳基因突變的患者均為中重型OI,且β-CTX(1.09±0.48ng/ml)和BMD Z值(腰椎-3.5±2.4)顯著高于Ⅰ型膠原基因變異的患者;80%存在嚴(yán)重骨骼畸形和活動(dòng)受限,但藍(lán)鞏膜(40%)和牙本質(zhì)發(fā)育不全(7%)少見。結(jié)論:Ⅰ型膠原編碼基因COLlA1和COLlA2是中國(guó)OI主要的致病基因,其次是常染色體顯性遺傳IFITM5基因,以及常染色體隱性遺傳SERPINF1和WNT1基因;不同臨床分型的OI患者具有顯著不同的基因突變譜。Ⅰ型膠原單倍劑量不足的患者臨床表型輕、骨骼畸形少;罕見常染色體隱性遺傳性O(shè)I患者病情嚴(yán)重、普遍有骨畸形和活動(dòng)受限,但缺乏典型骨骼外表現(xiàn)。此外,新型靶向二代測(cè)序平臺(tái)顯著提高了 OI分子診斷水平,為未來產(chǎn)前診斷和分子靶向治療奠定了初步基礎(chǔ)。第二部分成骨不全癥成人患者的臨床特點(diǎn)和雙膦酸鹽療效分析目的:成骨不全癥(Osteogenesis imperfecta,OI)是以骨骼脆性增加和反復(fù)骨折為特征的單基因遺傳性骨病。OI臨床異質(zhì)性大,可表現(xiàn)骨折風(fēng)險(xiǎn)輕度增加甚至圍生期死亡(Ⅰ-Ⅳ型);雙膦酸鹽(Bisphosphonates,BPs)是目前廣泛應(yīng)用的OI治療藥物,以阿侖膦酸鈉和唑來膦酸較為常用。然而,成人OI患者的具體臨床表現(xiàn)、是否仍需藥物干預(yù),尚不十分清楚。本研究探討成人OI患者的臨床特點(diǎn),并評(píng)估阿侖膦酸鈉和唑來膦酸治療成人OI的有效性和安全性。方法:納入2007-2016年于北京協(xié)和醫(yī)院內(nèi)分泌科首診的102例18歲以上OI患者�；仡櫺苑治龀扇薕I的臨床特點(diǎn),包括骨密度(bone mineral density,BMD)、骨轉(zhuǎn)換生化指標(biāo)血堿性磷酸酶(alkaline phosphatase,ALP)和β膠原降解產(chǎn)物(cross-linked C-telopeptide of type Ⅰ collagen,β-CTX)、25 羥維生素 D(25-hydroxyvitaminD,250HD)、甲狀旁腺激素(parathyroid hormone,PTH)、骨折率、影像學(xué)和骨骼外表現(xiàn),并與102例年齡、性別和體重指數(shù)匹配的健康對(duì)照者進(jìn)行比較。對(duì)2011-2015年接受治療的60例成人OI患者,按2:1分別納入阿侖膦酸鈉組(口服70mg/周)或唑來膦酸組(靜脈注射5mg/年),進(jìn)行為期2年的前瞻性藥物療效觀察;治療有效性指標(biāo)包括腰椎和髖部BMD變化率、骨轉(zhuǎn)換指標(biāo)變化率和新發(fā)骨折率。結(jié)果:102例成人OI患者中,近3/4為Ⅰ-Ⅳ型OI。與健康對(duì)照組相比,輕、中、重型(Ⅰ/Ⅲ/Ⅳ)患者的血ALP水平更高,僅重型(Ⅲ)患者β-CTX高于對(duì)照組(P=0.023)。成人OI患者250HD水平為16.2 ± 8.1 ng/ml,維生素D缺乏和不足分別占73.4%、89.0%。骨骼表型方面,成人OI的腰椎和髖部BMD明顯低于對(duì)照組,且Ⅰ/Ⅲ/Ⅳ型之間有統(tǒng)計(jì)學(xué)差異(P均0.001);此外,69.5%患者BMDZ值在-2SD以下。所有Ⅲ型患者均表現(xiàn)四肢長(zhǎng)骨畸形、脊柱側(cè)彎和壓縮性骨折,64%有牙本質(zhì)發(fā)育不全。成人OI的治療方面,共52例患者完成2年BPs治療和隨訪。經(jīng)2年治療,腰椎、股骨頸和全髖BMD在阿侖膦酸鈉組(分別為10.5、13.2、14.7%)和唑來膦酸組(分別為11.3、13.7、11.7%)顯著升高,且兩組間無顯著性差異(P均＞0.05);雙膦酸鹽均能顯著降低兩組骨轉(zhuǎn)換指標(biāo)ALP和β-CTX水平,組間無顯著性差異(P=0.12、0.48);與OI患者治療前的骨折率相比,治療后兩組的新發(fā)骨折率下降。結(jié)論:與健康人群相比,成人OI患者維生素D營(yíng)養(yǎng)狀況差、骨轉(zhuǎn)換水平高、腰椎和髖部骨密度低,仍存在高骨折風(fēng)險(xiǎn),故OI患者進(jìn)入成年期仍需要積極的藥物干預(yù)�？诜鲮⑺徕c和靜脈唑來膦酸均能有效提高成人OI患者的骨密度、抑制骨轉(zhuǎn)換,并可能降低骨折率,且兩種藥物的安全性較好。
[Abstract]:The first part of the gene mutation spectrum of Chinese large sample osteogenesis imperfecta: Osteogenesis Imperfecta (OI) is a group of genetic and phenotypic heterogenetic monogenic osteopathy, which is characterized by reduced bone mass, increased bone fragility and repeated fractures. Currently, the pathogenicity and potential molecular mechanisms of O1 in China are yet to be found. This study uses a new molecular diagnostic platform to explore the pathogenetic mutation spectrum of OI patients with large samples in China and to analyze the relationship between genotype and clinical phenotype in a variety of O1 patients with genetic patterns. Methods: This study uses the independent design, including the two generation of 19 OI pathogenicity genes. Sequence platform, 152 cases of OI patients who had been diagnosed in Peking Union Medical College Hospital for 2013-2016 years were tested for mutation of pathogenic gene. Sanger sequencing was used to test the families of each patient. In addition, a total of 261 cases of OI patients with previous gene detection in the group of O1 were included, and the patients with different genotypes were analyzed. Height, fracture rate, lumbar and femoral neck bone density (bone mineral density, BMD), bone conversion biochemical indicators of blood alkaline phosphatase (alkaline phosphatase, ALP) and beta collagen degradation products (cross-linked C-telopeptide of type I), bone malformation, and exoskeletal manifestations, and the common type I collagen gene variation In this study, two generation sequencing combined with family Sanger sequencing was used to detect the mutation of the pathogenic gene in 140 patients with OI, and 50 new mutation sites were detected. The detection rate of the new molecular diagnostic platform for OI was 92%, and the accuracy was 100%. in 261 cases of OI patients in this group. The gene (COLlA1/COLlA2) mutation was the most common (198 cases, 76%), followed by the IFITM5 gene (15 cases, 6%), SERPINF and WNT1 (12 cases, 4.6%) were two pathogenic genes in the autosomal recessive genetic OI. However, in the light OI, the detection rate of type I collagen gene was 84%, and in the medium heavy OI, the detection rate of type I collagen gene was only 68%, but recessive, but recessive The genetic variation of 21%. was compared with the structural variation of type I collagen. The height Z value (-0.4 + 1.3) and BMDZ value (-1.7 + 1.3, -2.7 + 2.2) of the patients with COL1A/ were higher, and the bone malformation was less (11%, P 0.05). Type I collagen alpha L chain structural variation site and blue sclera, dentin development, alpha 2 chain change point and BMD Z of femur neck There was a correlation between the values, but no correlation between the amino acid replacement type and the phenotype. All the patients with autosomal recessive gene mutations were medium heavy OI, and the values of beta -CTX (1.09 + 0.48ng/ml) and BMD Z (-3.5 + 2.4) were significantly higher than those of type I collagen gene mutation; 80% had severe skeletal deformity and limited activity, but blue was blue. Sclera (40%) and dentin dysplasia (7%) are rare. Conclusion: type I collagen encoding gene COLlA1 and COLlA2 are the main pathogenic genes of Chinese OI, followed by autosomal dominant genetic IFITM5 gene, and autosomal recessive SERPINF1 and WNT1 gene, and different genotyping of OI patients with significant different gene mutation spectrum. Patients with primary undoubled doses were mild in clinical phenotypes and less skeletal deformities; rare autosomal recessive OI patients were seriously ill, with common bone malformation and limited activity, but lack of typical exoskeletal manifestations. In addition, the new target two generation sequencing platform significantly improved the level of OI diagnosis for the future prenatal diagnosis and molecular targeting therapy. The clinical characteristics of second adult patients with osteogenesis imperfecta and analysis of the efficacy of bisphosphonates: Osteogenesis imperfecta (OI) is a single gene hereditary bone disease characterized by increased bone fragility and repeated fractures, with a large clinical heterogeneity of.OI, which may show a slight increase in the risk of fracture and even perinatal period. Death (type I - IV); Bisphosphonates (BPs) is currently a widely used OI treatment, with alendronate and zoledronic acid more commonly used. However, it is not clear whether the specific clinical manifestations of adult OI patients still need drug intervention. This study explored the clinical characteristics of adult OI patients and assessed alendronate and azole. Methylene phosphonate in the treatment of adult OI in 102 patients over 18 years old in Department of Endocrinology, Peking Union Medical College Hospital, for 2007-2016 years. The clinical characteristics of adult OI were analyzed retrospectively, including bone mineral density (bone mineral density, BMD), bone conversion biochemical markers of serum alkaline phosphatase (alkaline phosphatase, ALP), and beta collagen. Degradation products (cross-linked C-telopeptide of type I collagen, beta -CTX), 25 hydroxyvitamin D (25-hydroxyvitaminD, 250HD), parathyroid hormone (parathyroid hormone, PTH), fracture rate, imaging and exoskeletal performance, and compared with 102 healthy controls matched by age, sex and body mass index. For 2011-2015 years 60 adult OI patients were treated with 2:1 in the alendronate group (orally 70mg/ weeks) or zoledronic acid group (intravenous 5mg/ years) for a 2 year prospective drug effect. The therapeutic effectiveness index included the change rate of lumbar and hip BMD, the change rate of bone conversion index and the rate of new fracture. Results: 102 cases of adult OI patients, nearly 3/4 The level of blood ALP in light, middle, and heavy (I / III / IV) patients was higher than that in the control group of type I - IV OI.. The level of beta -CTX in severe (III) patients was higher than that of the control group (P=0.023). The level of 250HD in adult OI was 16.2 + 8.1 ng/ml, vitamin D deficiency and deficiency accounted for 73.4%, and 89.0%. skeletal phenotype in adult OI was significantly lower in the lumbar and hip than in adult OI. In the control group, there was a statistical difference between type I / III / IV (P 0.001); in addition, 69.5% patients had a BMDZ value below -2SD. All type III patients showed long bone malformation, scoliosis and compression fracture, 64% with dentin dysplasia. In adult OI treatment, a total of 52 patients completed 2 years of BPs treatment and follow-up. After 2 years of treatment, lumbar, femoral, femoral The bone neck and total hip BMD were significantly higher in the alendronate group (10.5,13.2,14.7%) and zoledronic acid group (respectively 11.3,13.7,11.7%), and there was no significant difference between the two groups (P > 0.05). The bisphosphonates could significantly reduce the level of ALP and beta -CTX in two groups of bone conversion indicators, and there was no significant difference between the groups (P=0.12,0.48); before the treatment of OI patients. Compared to the two groups, the new fracture rate in the two groups decreased. Conclusion: compared with healthy people, adult OI patients have poor vitamin D nutritional status, high bone conversion level, low bone density in the lumbar and hip and high fracture risk, so OI patients still need active drug intervention to enter adulthood. Oral alendronate and zoledronic acid are both oral. It can effectively increase bone mineral density in adult OI patients, inhibit bone turnover and reduce fracture rate, and the safety of the two drugs is better.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R681.1

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