本文選題：青少年特發(fā)性脊柱側(cè)凸 + 生長發(fā)育高峰�。� 參考：《南京大學(xué)》2014年博士論文

【摘要】：第一章(第一節(jié))生長發(fā)育高峰關(guān)聯(lián)基因與青少年特發(fā)性脊柱側(cè)凸易感性及進(jìn)展的相關(guān)性研究 目的：探討人體生長發(fā)育高峰(PHV)關(guān)聯(lián)基因與漢族青少年特發(fā)性脊柱側(cè)凸(AIS)的發(fā)生及進(jìn)展的相關(guān)性。 方法：選擇9個與PHV、生長高峰年齡或成年身高相關(guān)基因作為AIS致病候選基因,包括SOCS2、SF3B、SV2A、C17orf67、CABLES1、DOT1L、CDK6, C6orf106和LIN28B。在我科DNA數(shù)據(jù)庫中選擇500例AIS患者和494例年齡匹配的健康對象進(jìn)行基因分型研究并作病例-對照關(guān)聯(lián)分析。使用熒光探針PCR技術(shù)對實驗對象進(jìn)行基因分型。通過卡方檢驗計算各個基因單核苷酸多態(tài)性(SNP)位點與AIS疾病發(fā)生的相關(guān)性及對應(yīng)風(fēng)險值。SPSS16.0軟件被用于統(tǒng)計分析。P值小于0.05被認(rèn)為有顯著性差異。 結(jié)果：DOT1L基因的rs12459350位點和C17orf67基因的rs4794665位點被發(fā)現(xiàn)與AIS易感性之間存在顯著關(guān)聯(lián)。含有PHV相關(guān)的rs12459350等位基因G和成年身高相關(guān)的rs4794665等位基因A人群的AIS易感性可顯著增加(rs12459350： P=0.001,風(fēng)險比=1.16；rs4794665：P=0.006,風(fēng)險比=1.33)。上述基因的SNP位點與AIS疾病進(jìn)展無顯著性關(guān)聯(lián)。 結(jié)論：C17orf67基因的SNP位點rs4794665和DOT1L基因的SNP位點rs12459350為AIS易感性位點。由于上述位點同時與青春期PHV發(fā)育相關(guān),證明了遺傳因素對AIS患者異常生長發(fā)育模式有顯著影響。 第一章(第二節(jié))利用全基因組關(guān)聯(lián)分析定位漢族人群AIS患者的致病基因 目的：對漢族青少年特發(fā)性脊柱側(cè)凸(AIS)患者行全基因組關(guān)聯(lián)分析研究(GWAS)以獲得易感基因位點,并探討AIS易感基因在疾病發(fā)病機制中的作用。 方法：從本科室漢族人群AIS患者DNA數(shù)據(jù)庫中選取了336例患者納入本次研究。入選標(biāo)注如下：1.女性患者；2.年齡大于18周歲；3.彎型為右主胸彎。594例性別匹配的健康人作為正常對照。用AFFX SNP6.0芯片進(jìn)行基因芯片掃描及全基因組關(guān)聯(lián)分析,篩選AIS陽性SNPs位點并進(jìn)一步分析易感基因相關(guān)的致病通路。EIGENSTRAT軟件,PLINK軟件及R軟件分別用于人群分層主成分析及病例-對照關(guān)聯(lián)分析。P值小于10-8被認(rèn)為有顯著差異。 結(jié)果：人群主成分析顯示病例及對照組均來自漢族人群,無明顯人群分層現(xiàn)象。經(jīng)質(zhì)量控制共有544,868個SNPs位點被納入病例-對照關(guān)聯(lián)分析。經(jīng)分析共篩選出的18個陽性SNPs位點與AIS疾病發(fā)生顯著相關(guān)。先前見諸報道的LBX1基因,CHL1基因及GPR126基因均不在本次發(fā)現(xiàn)的位點連鎖區(qū)域內(nèi)。 結(jié)論：本GWAS研究發(fā)現(xiàn)了新的漢族人群AIS致病基因,同時該基因與日本人群及白種人群的GWAS研究發(fā)現(xiàn)有顯著差異,證明了AIS遺傳病因?qū)W研究存在著顯著的種族差異。上述基因的發(fā)現(xiàn)為今后進(jìn)一步研究漢族AIS患者的發(fā)病機制起重要作用。 第二章(第一節(jié))建立預(yù)測漢族AIS患者支具療效的Logistic回歸模型 目的：研究可導(dǎo)致青少年特發(fā)性脊柱側(cè)凸患者疾病發(fā)展的獨立危險因素,同時通過建立logistic回歸模型以預(yù)測支具療效。 方法：所有實驗對象均為自2005年1月至2008年12月在鼓樓醫(yī)院脊柱外科門診行規(guī)范化支具治療的AIS患者。根據(jù)患者側(cè)凸類型選擇相應(yīng)的支具進(jìn)行治療。記錄初次就診及末次隨訪的原發(fā)彎的Cobb角。疾病進(jìn)展的標(biāo)準(zhǔn)為原發(fā)彎增加5。。分別選取雌激素受體a(Estrogen receptor a, ERa),雌激素受體3(Estrogen receptorβ, ERβ),色氨酸羥化酶1(Tryptophan Hydroxylase1,TPH-1),褪黑素受體1B(melatonin receptor1B, MTNR1B)和matrillin-1(MATN1)等易感基因的單核苷酸多態(tài)性位點(single nucleotide polymorphism, SNPs)進(jìn)行PCR-RFLP基因分型。根據(jù)支具治療效果將患者分為進(jìn)展組和穩(wěn)定組兩組,行t檢驗或χ2檢驗比較兩組之間的初診年齡、Risser征、側(cè)凸類型、初診Cobb角及基因型分布的差異。將p0.05的變量納入logistic回歸分析,確定影響側(cè)彎進(jìn)展的獨立因素。同時建立回歸模型,對AIS患者側(cè)彎進(jìn)展進(jìn)行預(yù)測。 結(jié)果：共計312名患者入選本研究。其中222位患者側(cè)彎穩(wěn)定,90位患者側(cè)彎有顯著進(jìn)展。側(cè)彎進(jìn)展組的患者Risser征顯著低于側(cè)彎穩(wěn)定組(0.8±1.2vs1.4±1.3,p0.001)。兩組患者的初診Cobb角和年齡無顯著差異。在不同的側(cè)凸類型中,胸腰雙主彎的支具治療失敗率最低,但與其他彎型無顯著差異。ERα基因型GA在側(cè)彎進(jìn)展組中比例顯著高于側(cè)彎穩(wěn)定組(50.9%vs17.9%,p0.001)。TPH-1基因型AT在側(cè)彎進(jìn)展組中比例亦顯著高于側(cè)彎穩(wěn)定組(33.3%vs13.0%,p=0.002)。Logistic回歸分析顯示初診Risser征(OR=2.289),ERa等位基因G(OR=3.559)及TPH-1等位基因A(OR=2.092)是影響側(cè)彎進(jìn)展的獨立危險因素。將三者納入回歸模型后顯示,當(dāng)診斷點設(shè)為0.5時,該模型的敏感性和特異性分別為41.7%和92.3%；若診斷點設(shè)為0.2,其敏感性可上升至86.7%,而特異性下降為30.3%。 討論：Risser征,ERa等位基因G和TPH-1等位基因A是AIS患者側(cè)彎進(jìn)展的危險因素。通過Logistic回歸模型,我們可初步判斷AIS患者支具治療成功與否。然而由于預(yù)測因子的不足,該模型總體預(yù)測能力尚低,并不足以應(yīng)用于臨床。為了進(jìn)一步完善模型的預(yù)測能力,更多的預(yù)測因子仍有待發(fā)掘。 第三章(第一節(jié))先天性脊柱骨骺發(fā)育不良伴脊柱側(cè)凸家系的致病基因檢測 目的：檢測先天性脊柱骨骺發(fā)育不良(SEDC)伴脊柱側(cè)凸的致病基因 方法：一個涉及3代5例SEDC患者的家系被納入研究。所有患者均接受全面的身體和影像學(xué)檢查。獲得每個參與者的知情同意書并收集患者和健康對照者的DNA樣本。利用兩個短串聯(lián)重復(fù)序列(STR)多態(tài)性標(biāo)記于COL2A1基因側(cè)翼區(qū)進(jìn)行檢測以確定每個病人的單倍型。隨即對COL2A1基因進(jìn)行序列分析以確定潛在的遺傳突變。 結(jié)果：單倍型分析表明,同一疾病相關(guān)的單倍型在整個家系呈共分離。通過D12S85和D12S368可得到最大LOD值為1.5。DNA序列分析揭示了在COL2A1基因的第25外顯子存在c.1636G/A變異。該546位點的甘氨酸密碼子GGT被轉(zhuǎn)換為編碼絲氨酸的密碼子AGT�；颊呔鶠殡s合的G546S突變,該突變未見于任一正常個體的或未受疾病影響的其他家族成員。 結(jié)論：本研究首次報告在SEDC家族COL2A1基因存在G546S突變。雖然在識別該疾病突變譜方面已有巨大成就,仍需更深入的研究以進(jìn)一步確定疾病基因型與表型之間的相關(guān)性。
[Abstract]:The correlation between the correlation gene of growth and development of the first chapter ( section I ) and the susceptibility and progression of adolescent idiopathic scoliosis

Objective : To investigate the correlation between the correlation gene of human growth and development ( PHV ) and the occurrence and progression of idiopathic scoliosis ( AIS ) in Han nationality .

Methods : Nine patients with PHV were selected as candidate genes of AIS , including SOCS2 , SF3B , SV2A , C17orf67 , CABLES1 , DOT1L , CDk6 , C6orf106 and LIN28B . In our DNA database , 500 AIS patients and 494 age - matched healthy subjects were genotyped .

Results : rs12459350 locus of DOT1L gene and rs4794665 locus of C17orf67 gene were found to be significantly associated with AIS susceptibility . The AIS susceptibility of rs12459350 allele G containing PHV and rs4794665 allele A in adult height was significantly increased ( rs12459350 : P = 0.001 , risk ratio = 1.16 ;
rs4794665 : P = 0.006 , hazard ratio = 1.33 ) . There was no significant association between SNP site of the above gene and AIS disease progression .

Conclusion : The SNP loci rs4794665 and rs12459350 of the C17orf67 gene are the susceptibility loci of AIS . Since the above - mentioned sites are related to the development of PHV in adolescence , it is shown that the genetic factors have a significant effect on the abnormal growth patterns of AIS patients .

Chapter 1 ( Section II ) Using Whole Genome Correlation Analysis to locate the pathogenic genes of AIS patients in Han population

Objective : To study the effect of AIS susceptibility gene on the pathogenesis of idiopathic scoliosis ( AIS ) patients with idiopathic scoliosis ( AIS ) .

Methods : 336 patients were selected from the DNA database of AIS patients in the Han nationality in the undergraduate chamber . The selection was as follows : 1 . Female patients ;
2 . The age is more than 18 years ;
3 . There were 594 gender - matched healthy individuals as normal controls . The gene chip scanning and genome - wide association analysis were performed by using the AFFX SNP6.0 chip , and the pathogenic pathways associated with susceptible genes were further analyzed . The EIGENSTRAT software , PLINK software and R software were used for the analysis of population stratification and the case - control correlation analysis respectively . The P value was less than 10 - 8 considered significant difference .

Results : The results showed that there were 544 and 868 SNPs in the study group , and there were 544 and 868 SNPs in the control group . The results showed that the 18 positive SNPs were associated with AIS disease .

Conclusion : The GWAS study has found that there is a significant difference in the etiology of AIS in Chinese Han population . The findings of these genes play an important role in the further study of the pathogenesis of AIS patients in the future .

Chapter 2 ( Section I ) Logistic regression model for predicting the curative effect of Chinese patients with AIS

Objective : To study the independent risk factors which can lead to the development of adolescent idiopathic scoliosis .

Methods : All the subjects were AIS patients treated with standardized brace from January 2005 to December 2008 . The treatment was performed according to the patient ' s scoliosis type . The primary and final follow - up were recorded . The incidence of disease progression was 5 . Single nucleotide polymorphism ( SNPs ) of susceptible genes such as estrogen receptor a ( ERa ) , estrogen receptor 尾 ( ER尾 ) , tryptophan hydroxylase 1 ( TPH - 1 ) , melatonin receptor 1B ( TPH - 1 ) , melatonin receptor 1B ( TPH - 1 ) , melatonin receptor 1B ( MTNR1B ) and matrixing - 1 ( MATN1 ) were selected .

Results : A total of 312 patients were enrolled in the study . Among them , 222 patients had stable lateral bending and 90 patients had significant progress . The patient Risser sign was significantly lower than that in the lateral bending stable group ( 0.8 鹵 1.2 v1.4 鹵 1.3 , p0.001 ) . There was no significant difference in the angle and age between the two groups . In the different scoliosis types , the treatment failure rate was the lowest among the two main groups , but there was no significant difference between the two groups . The proportion of ER偽 gene GA in the lateral bending progression group was significantly higher than that in the lateral bending stable group ( 50.9 % vs 17.9 % , p0.001 ) . The proportion of TPH - 1 genotype AT was significantly higher in the lateral bending progression group than that in the lateral group ( 33.3 % vs 13.0 % , p = 0.002 ) . Logistic regression analysis showed that Risser sign ( OR = 2.289 ) , ERa allele G ( OR = 3.559 ) and TPH - 1 allele A ( OR = 2.092 ) were independent risk factors affecting the progress of lateral bending .
If the diagnostic point is set to 0.2 , its sensitivity can be increased to 86.7 % , and the specificity is reduced to 30.3 % .

Conclusion : Risser sign , ERa allele G and TPH - 1 allele A are the risk factors of lateral bending progress in AIS patients . Through Logistic regression model , we can judge whether the treatment of AIS patients is successful or not . However , due to the shortage of predictors , the overall predictive ability of the model is still low , which is not enough to be applied to clinical . In order to further improve the forecasting ability of the model , more prediction factors still need to be explored .

Chapter III ( Section I ) Pathogenic gene detection of congenital scoliosis with scoliosis

Objective : To detect the pathogenesis of congenital scoliosis ( SEDC ) with scoliosis

Method : A family of 5 SEDC patients involved in the study was included in the study . All patients received a comprehensive physical and radiological examination . Each participant ' s informed consent was obtained and DNA samples from patients and healthy controls were collected . The COL2A1 gene flanking region was detected using two short tandem repeat sequences ( STR ) polymorphisms to determine the haplotype of each patient . The COL2A1 gene was then subjected to sequence analysis to identify potential genetic mutations .

Results : Single - fold analysis showed that the haplotype associated with the same disease was isolated from the whole family . The maximum LOD value was 1.5 . DNA sequence analysis revealed that there was c . 1636G / A mutation in exon 25 of COL2A1 gene . The glycine codon GGT at 546 site was converted to codons encoding serine . All patients were heterozygous G546S mutations , which were not found in any other family member of normal individuals or unaffected by the disease .

Conclusion : The G546S mutation in the SEDC family COL2A1 gene is reported for the first time . Although there has been a great achievement in identifying the mutation spectrum of the disease , more in - depth study is needed to further determine the correlation between disease gene type and phenotype .
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R682.3

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相關(guān)期刊論文 前1條

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