汉族青少年特发性脊柱侧凸致病基因的定位及相关临床应用

发布时间：2018-06-01 03:49

本文选题：青少年特发性脊柱侧凸 + 生长发育高峰　；参考：《南京大学》2014年博士论文

【摘要】：第一章(第一节)生长发育高峰关联基因与青少年特发性脊柱侧凸易感性及进展的相关性研究目的：探讨人体生长发育高峰(PHV)关联基因与汉族青少年特发性脊柱侧凸(AIS)的发生及进展的相关性。方法：选择9个与PHV、生长高峰年龄或成年身高相关基因作为AIS致病候选基因,包括SOCS2、SF3B、SV2A、C17orf67、CABLES1、DOT1L、CDK6, C6orf106和LIN28B。在我科DNA数据库中选择500例AIS患者和494例年龄匹配的健康对象进行基因分型研究并作病例-对照关联分析。使用荧光探针PCR技术对实验对象进行基因分型。通过卡方检验计算各个基因单核苷酸多态性(SNP)位点与AIS疾病发生的相关性及对应风险值。SPSS16.0软件被用于统计分析。P值小于0.05被认为有显著性差异。结果：DOT1L基因的rs12459350位点和C17orf67基因的rs4794665位点被发现与AIS易感性之间存在显著关联。含有PHV相关的rs12459350等位基因G和成年身高相关的rs4794665等位基因A人群的AIS易感性可显著增加(rs12459350： P=0.001,风险比=1.16；rs4794665：P=0.006,风险比=1.33)。上述基因的SNP位点与AIS疾病进展无显著性关联。结论：C17orf67基因的SNP位点rs4794665和DOT1L基因的SNP位点rs12459350为AIS易感性位点。由于上述位点同时与青春期PHV发育相关,证明了遗传因素对AIS患者异常生长发育模式有显著影响。第一章(第二节)利用全基因组关联分析定位汉族人群AIS患者的致病基因目的：对汉族青少年特发性脊柱侧凸(AIS)患者行全基因组关联分析研究(GWAS)以获得易感基因位点,并探讨AIS易感基因在疾病发病机制中的作用。方法：从本科室汉族人群AIS患者DNA数据库中选取了336例患者纳入本次研究。入选标注如下：1.女性患者；2.年龄大于18周岁；3.弯型为右主胸弯。594例性别匹配的健康人作为正常对照。用AFFX SNP6.0芯片进行基因芯片扫描及全基因组关联分析,筛选AIS阳性SNPs位点并进一步分析易感基因相关的致病通路。EIGENSTRAT软件,PLINK软件及R软件分别用于人群分层主成分析及病例-对照关联分析。P值小于10-8被认为有显著差异。结果：人群主成分析显示病例及对照组均来自汉族人群,无明显人群分层现象。经质量控制共有544,868个SNPs位点被纳入病例-对照关联分析。经分析共筛选出的18个阳性SNPs位点与AIS疾病发生显著相关。先前见诸报道的LBX1基因,CHL1基因及GPR126基因均不在本次发现的位点连锁区域内。结论：本GWAS研究发现了新的汉族人群AIS致病基因,同时该基因与日本人群及白种人群的GWAS研究发现有显著差异,证明了AIS遗传病因学研究存在着显著的种族差异。上述基因的发现为今后进一步研究汉族AIS患者的发病机制起重要作用。第二章(第一节)建立预测汉族AIS患者支具疗效的Logistic回归模型目的：研究可导致青少年特发性脊柱侧凸患者疾病发展的独立危险因素,同时通过建立logistic回归模型以预测支具疗效。方法：所有实验对象均为自2005年1月至2008年12月在鼓楼医院脊柱外科门诊行规范化支具治疗的AIS患者。根据患者侧凸类型选择相应的支具进行治疗。记录初次就诊及末次随访的原发弯的Cobb角。疾病进展的标准为原发弯增加5。。分别选取雌激素受体a(Estrogen receptor a, ERa),雌激素受体3(Estrogen receptorβ, ERβ),色氨酸羟化酶1(Tryptophan Hydroxylase1,TPH-1),褪黑素受体1B(melatonin receptor1B, MTNR1B)和matrillin-1(MATN1)等易感基因的单核苷酸多态性位点(single nucleotide polymorphism, SNPs)进行PCR-RFLP基因分型。根据支具治疗效果将患者分为进展组和稳定组两组,行t检验或χ2检验比较两组之间的初诊年龄、Risser征、侧凸类型、初诊Cobb角及基因型分布的差异。将p0.05的变量纳入logistic回归分析,确定影响侧弯进展的独立因素。同时建立回归模型,对AIS患者侧弯进展进行预测。结果：共计312名患者入选本研究。其中222位患者侧弯稳定,90位患者侧弯有显著进展。侧弯进展组的患者Risser征显著低于侧弯稳定组(0.8±1.2vs1.4±1.3,p0.001)。两组患者的初诊Cobb角和年龄无显著差异。在不同的侧凸类型中,胸腰双主弯的支具治疗失败率最低,但与其他弯型无显著差异。ERα基因型GA在侧弯进展组中比例显著高于侧弯稳定组(50.9%vs17.9%,p0.001)。TPH-1基因型AT在侧弯进展组中比例亦显著高于侧弯稳定组(33.3%vs13.0%,p=0.002)。Logistic回归分析显示初诊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)是影响侧弯进展的独立危险因素。将三者纳入回归模型后显示,当诊断点设为0.5时,该模型的敏感性和特异性分别为41.7%和92.3%；若诊断点设为0.2,其敏感性可上升至86.7%,而特异性下降为30.3%。讨论：Risser征,ERa等位基因G和TPH-1等位基因A是AIS患者侧弯进展的危险因素。通过Logistic回归模型,我们可初步判断AIS患者支具治疗成功与否。然而由于预测因子的不足,该模型总体预测能力尚低,并不足以应用于临床。为了进一步完善模型的预测能力,更多的预测因子仍有待发掘。第三章(第一节)先天性脊柱骨骺发育不良伴脊柱侧凸家系的致病基因检测目的：检测先天性脊柱骨骺发育不良(SEDC)伴脊柱侧凸的致病基因方法：一个涉及3代5例SEDC患者的家系被纳入研究。所有患者均接受全面的身体和影像学检查。获得每个参与者的知情同意书并收集患者和健康对照者的DNA样本。利用两个短串联重复序列(STR)多态性标记于COL2A1基因侧翼区进行检测以确定每个病人的单倍型。随即对COL2A1基因进行序列分析以确定潜在的遗传突变。结果：单倍型分析表明,同一疾病相关的单倍型在整个家系呈共分离。通过D12S85和D12S368可得到最大LOD值为1.5。DNA序列分析揭示了在COL2A1基因的第25外显子存在c.1636G/A变异。该546位点的甘氨酸密码子GGT被转换为编码丝氨酸的密码子AGT。患者均为杂合的G546S突变,该突变未见于任一正常个体的或未受疾病影响的其他家族成员。结论：本研究首次报告在SEDC家族COL2A1基因存在G546S突变。虽然在识别该疾病突变谱方面已有巨大成就,仍需更深入的研究以进一步确定疾病基因型与表型之间的相关性。
[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 卤 1.2 v1.4 卤 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伪 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 .
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R682.3

【参考文献】