摘要
研究背景
青少年特发性脊柱侧凸(Adolescent idiopathic scoliosis, AIS)是青少年常见的脊柱畸形,发病年龄在10～20岁之间,人群中发病率约1.5-3%。尽管目前对AIS的发病原因和具体机制尚不清楚,但近年来遗传学和分子生物学的进步,以及对AIS的遗传病因学研究逐步深入,发现它是一种多基因相关的遗传性疾病。
对于多基因相关的遗传性疾病,常用的研究方法有关联分析、单倍体型分析法、候选基因法。因其能够简便、直接、准确地阐述疾病相关基因的关联性和作用机制,目前已将其作为群体遗传学研究和疾病相关基因检测的重要手段。
目前有多种指导手术治疗方法选择的AIS临床分型,尚无从不同的临床表型角度分组分层对AIS遗传病因学机制进行研究的相关报道。因此有必要通过统计学的分析取得对临床分型在基因层面的关联性。
研究目的
1.通过对钙调蛋白1基因(Calmodulin1, CALM1基因)、钙调蛋白2基因(Calmodulin2, CALM2基因)与AIS之间的关联性分析,明确其在AIS发病机制中发挥的效应。
2.通过对CALM1基因及CALM2基因SNPs位点等位基因和基因型与AIS临床表型的关联分析,在基因水平上探索CALM1基因及CALM2基因与AIS不同临床表型(PUMC分型)之间的关系,从遗传学上丰富AIS临床表型的内容。
研究方法
1.研究对象：选取2006年8月～2008年5月期间北京协和医院骨科收治并已确诊的汉族青少年特发性脊柱侧凸患者,共107例,其中男性17例,女性90例,平均年龄15.53岁(10-20岁)。对照组则为同期北京协和医院收治的感染或创伤患者、健康查体者等汉族患者,共107例,男性17例,女性90例,平均年龄15.12岁(10-20岁)。
2.研究方法：病例-对照研究。
2.1根据国际人类基因组单倍体型图计划(http://www.hapmap.org/)提供的基因型数据,应用Haploview4.0软件选取CALM1基因/CALM2基因的SNPs位点,优先选取杂合度高于10%,位于外显子区域或有错义突变的SNPs和位于3’-、5’-调控区的SNPs作为遗传标记、共选取21个SNPs;
2.2分别按照侧凸主弯顶点所在位置、PUMC分型对病例组进行分组；
2.3QIAamp(?) DNA Blood Mini Kit试剂盒提取DNA;
2.4所有研究对象应用Illumina公司VeraCode GoldenGate Genotyping Assay对所选的SNPs进行基因型鉴定；
2.5拟合优度x2检验和Fisher双侧精确检验分析病例、对照组基因型频率的分布是否符合H-W平衡；
2.6基于基因型/等位基因频率的“病例-对照”关联分析；
2.7在基因型/等位基因频率关联分析基础上,应用在线软件(SNPstats)进行非条件Logistic回归模型评估单位点基因型与AIS发生风险的相关程度；
2.8连锁不平衡和单倍体型分析：应用Haploview4.0软件计算CALM1基因/CALM2基因所有单倍体型在病例组和对照组之间的分布差异；
2.9基于基因型/等位基因频率的病例组不同临床表型之间的关联分析。
研究结果
1.流行病学资料分析：病例组与对照组的性别、年龄分布无统计学差异。
2.我们共检测了两个基因的21个位点,最终有2个位点因为检出率较低被放弃,其余19个位点检出率均>98%。这19个SNPs位点均具有多态性,对照组中等位基因和基因型符合Hardy-Weinberg平衡。
3. SNPs等位基因多态性：rs10153674等位基因在病例组和对照组中的分布频率统计学上存在显著差异(p=0.003),rs10153674位点等位基因G与AIS的易感性升高有关。
4.基因型多态性4.1rs10153674基因型在病例组和对照组中的分布频率统计学上存在显著差异(p=0.006),rs10153674基因型G/G与AIS的易感性升高有关。在非条件Logistic回归分析中,经年龄和性别校正后,rs10153674位点符合Dominant遗传模型(OR=2.75,95％CI:1.36-5.55,p=0.0035,AIC=294.8),及rs1027478位点符合Overdominant遗传模型(OR=1.00,p=0.025, AIC=298.3).
4.2单倍体：在多位点单倍体型分析中,发现1种阳性单倍体型：SNP18G—SNP13A—SNP14A—SNP16A—SNP20A—SNP19A.
4.3结合PUMC分型,rs10153674基因型及rs1027478基因型在PUMC Ⅰ型和对照组中的分布频率统计学上存在显著差异(P=0.038和P=0.006),进一步分析得知rs10153674基因型G/G.rs1027478基因型A/A与PUMC Ⅰ型的发生有关。
4.4结合AIS主弯顶点位置分析,rs10153674基因型在胸弯和对照组中的分布频率统计学上存在显著差异(P=0.047),进一步分析得知rs10153674基因型G/G与胸弯的发生有关。
4.519个SNPs位点基因型与病例组主侧弯Cobb's角度数之间无统计学差异。
结论
1.钙调蛋白2基因SNP位点多态性与青少年特发性脊柱侧凸(AIS)发生相关,可能是AIS的易感基因之一。
2.PUMC Ⅰ型AIS的发生可能与rs10153674和rs1027478基因型多态性有关；胸弯AIS的发生可能和rs10153674基因型多态性有关。
3.钙调蛋白2基因不同SNP位点等位基因多态性可能和特发性脊柱侧凸不同临床亚型相关联。研究初步显示AIS的PUMC分型可能和调蛋白2基因不同的SNP位点多态性有关,提示PUMC分型不仅是合理的临床分型,同时也可能符合遗传病因学机制。
Background
Adolescent idiopathic scoliosis (AIS) is the most common pediatric spinal deformity affecting1.5%to3%of the population, it usually occurs at the age of10to20years old. The cause of AIS is still not clear, but with the rapid development of genetics and molecular biology in recent years, genetics researches of AIS are brought into focus gradually. Recent studies show that AIS is a multifactorial inheritance disease.
Candidate gene analysis, association analysis and haplotype analysis are widely used in investigating multigenetic disease. They are direct and effective means of elucidating the mechanism of disease-related gene.
There are several clinical phenotypes classification for AIS, but there are no report about the association of genetic mechanism with different clinical classifications. Thus it is necessary to do some studis to analyse the association of genotypes and clinical classifications of AIS.
Objects
1. To investigat the relationship between CALM1/CALM2gene and AIS, and to investigate the major effect of CALM1/CALM2Gene in AIS patients,
2. To investigat the relationship between the genotypes of SNPs and the clinical phenotypes of AIS (such as PUMC classification).
Methods
We enrolled a total of107patients (17males,90females, mean age15.53y/o) diagnosed with adolescent idiopathic scoliosis admitted in Peking Union Medical College Hospital (PUMCH) in this study according to inclusion and exclusion criteria between August2006and May2008. The control group also comprised107patients (7males,90females, mean age15.12y/o) and consisted of infection(57), inflammatory disease(28),trauma(17), and others(5) at PUMCH during the same period. It was a hospital-based case-ontrol design. All the control subjects were frequency-matched to the cases on age (±3years), gender and the Han nationality. We recorded PUMC classification, curve pattern, cobb's angle of main curve of AIS patients. We extracted genomic DNA from peripheral blood leukocytes with QIAamp DNA Blood Mini Kit. Based on genotype data from the international HapMap project,12single nucleotide polymorphisms (SNPs) in calmodulin1(CALM1) gene and9single nucleotide polymorphisms (SNPs) in calmodulin2(CALM2) gene were initially choosed by Haploview4.0software. Genotyping of all selected SNPs was done by SNPstream technolofy (Beckman Coulter SNPstream). Haplotype frequencies were estimated and difference in haplotype distributions between cases and controls were assessed by Haploview4.0software. We use the SNPstats, a online software, to analyse all the data of21SNPs with polymorphism by the association analysis based on alleles and phenotypes of SNPs and95%confidence intervals(CIs) were computed by the unconditional logistic regression to estimate the relative risk for the single locus genotypes.
Results
1. Analysis of Epidemiologic data:there were no significant difference of age distribution and sex proportion between case group and control group.
2. We failed in composing premier of two SNPs (rs12885713,rs5871) and abandoned them in this study.The remained19SNPs were genotyped and all polymorphisms were in Hardy-Weinberg equilibrium of contrl group.
3. SNP Allele:the allele frequency distributions of rs10153674was statistically different between case group and control group(P=0.003respectively).
4. SNP genotype4.1The genotypes distributions of rs10153674was statistically different between caseg roup and control group(P=0.006respectively). In the unconditional logistic regression analysis,after adjustment for age and gender, rs10153674and rs1027478both showed significant difference in Dominant model (OR=2.75,95%CI=1.36-5.55, p=0.0035, AIC=294.8), Overdominant model (OR=1.00, p=0.025, AIC=298.3).
4.2Haplotype:we got1positive haplotype:SNP18G-SNP13A—SNP14A-SNP16A-SNP20A-SNP19A.
4.3The genotypes distributions of rs10153674and rs1027478were statistically different between PUMC I AIS group and control group (P=0.038,0.006respectively).
4.4The genotype distributions of rs10153674was statistically different between thoracic curve group and control group (P=0.047respectively).
4.5There was no statistical difference between main curve severity and genotypes distributions of all of19SNPs.
Conclusion
1. Different SNPs of CALM2gene are associated with AIS and may play a role in the development of AIS.
2. The development of PUMC I AIS might be related to genotype polymorphisms of rs10153674and rs1027478. The development of thoracic curve of AIS might be related to genotype polymorphisms of rs10153674.
3. The different clinical phenotype of AIS might be related to the different SNP site. It was preliminarily suggested that PUMC classification of AIS was not only the clinical classification, but also the logical combination of genetic mechanism and phenotype.
引文
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