| Chapter1(sectionl) Association between genetic determinants of peak height velocity during puberty and predisposition to adolescent idiopathic scoliosisObjective. To investigate whether the genetic determinants of timing and magnitude of PHV during puberty are associated with the susceptibility or curve progression of the female AIS.Methods. A gene-based association study was conducted using9SNPs in or near SOCS2, SF3B4/SV2A, C17orf67, CABLES1, DOT1L, CDK6, C6orf106, and LIN28B with confirmed association with PHV, peak growth age, or adult height. A total of500patients with AIS and494age-matched healthy controls were genotyped using the PCR-based Invader assay. Case-control study and case only study were performed to define the contribution of the9SNPs to predisposition and curve severity of AIS.Results. Strong associations between rs12459350in DOT1L, rs4794665in C17orf67, and susceptibility of AIS were found, with the PHV increasing allele G of rs12459350 and PHV/adult height increasing allele A of rs4794665both being significant predisposition alleles of AIS (P=0.001for rs12459350, odds ratio=1.16,95%confi dence interval=1.06-1.27; P=0.006for rs4794665, odd ratio=1.33,95confi dence interval=1.09-1.62). None of the genotyped SNPs was associated with curve severity in patients with AIS.Conclusion. Polymorphisms of the rs4794665in C17orf67and rs12459350in DOT1L were associated with combined predisposition to AIS susceptibility and higher pubertal PHV, which strongly mirrored the anthropometric fi ndings of taller pubertal stature and accelerated growth rate described in AIS. Chapter1(section2) Genome-wide association study in adolescent idiopathic scoliosis patients of Chinese Han populationObjective To investigate the pathogenesis of adolescent idiopathic scoliosis (AIS) in Chinese Han population using genome-wide association study (GWAS)Methods336AIS patients were enrolled in the study according to the following inclusion criteria:1.female;2.aged more than18years old;3.with major thoracic curve.594gender-matched normal subjects were enrolled as control. Blood samples were collected from all the subjects with informed consent signed. Affx SNP6.0chips with920,000SNPs were used for genotyping. Quality control of the chips was performed using EIGENSTRAT. Principal component analysis (PCA) was used to determine potential population stratification. PLINK software package was applied to case-control association analysis, so as to determine SNPs significantly associated with the occurrence of AIS. A p value of10-8was considered as genome significance.Results The mean call rate of the chips was99.5%. PCA showed that all the cases and control were clustered in the Han population. The genome inflation factor was1.045, indicating there was no remarkable population stratification among the subjects. After quality control,544,868SNPs were finally included in the case-control association analysis.18SNPs were found to have a p value of less than10"8, strongly suggesting their role in the occurrence of AIS. The3genes reported by previous GWAS studies, including LBX1, CHL1and GPR126, were not within the linkage region of the above-mentioned SNPs.Conclusion New AIS-related genes were found in this study with remarkable genome significance, which will serve as a fundamental work for future study on the pathogenesis of AIS. Chapter2(sectionl) Application of logistic regression model to the prediction of bracing outcome in patients with adolescent idiopathic scoliosisObjectives To develop and validate a logistic regression model to predict bracing outcome in patients with adolescent idiopathic scoliosis (AIS).Methods312AIS patients prescribed with brace treatment were enrolled in our study. All the patients were divided into two groups (curve progression/non-curve progression) according to the outcome of brace treatment. The curve progression was defined as a curve progression of more than5degree from the initial evaluation or as surgical intervention. The differences of age, Risser sign, initial curve magnitude and curve pattern between the two groups were compared. Meanwhile, we also investigated the differences of genotype and allele distribution of five single nucleotide polymorphism (SNP) sites between these two groups, including Estrogen receptor a (ERa), Estrogen receptor β (ERβ), Tryptophan Hydroxylase1(TPH-1), melatonin receptor1B (MTNR1B) and matrillin-1(MATN1) genes. Subsequently, a logistic regression model which included all the positive factors confirmed in the prior analysis was created to make a prediction of the curve progression.Results There were90cases (28.8%) in curve progression group and222cases (71.2%) in non-curve progression group. The mean value of Risser sign of the curve progression group was significantly lower than that of non-curve progression group. Moreover, patients in curve progression group had more genotype GA (50.9%VS17.9%, p<0.001) and allele G (27.1%VS12.0%, p<0.001) in SNP rs9340799site of ERa gene. Similarly, they also had more genotype AT (33.3%VS13.0%, p=0.002) and allele A (16.7%VS9.6%, p=0.033) in SNP rs10488682of TPH-1gene. Finally, Risser sign and genotype of ERa and TPH-1were entered into the logistic regression model. With the cut-off point set at0.50, the sensitivity and specificity of the model were41.7%and92.3%, respectively. When the cut-off point was set at0.2, the sensitivity of the model rose to86.7%, while the specificity of the model dropped to30.3%.Conclusion Risser sign and genotype of ERa and TPH-1were independent factors predictive of curve progression. With current model, we made a preliminary step towards the prediction of the bracing outcome. However, the model was not powerful enough for its application to the clinical practice. To clarify which kind of patients may have failure outcome of bracing outcome, further investigation for more predictors should be carried out. Chapter3(section1) A novel mutation in COL2A1leading to spondyloepiphyseal dysplasia congenita in a three-generation familyObjective To investigate the genotype of COL2A1in a three-generation spondylo-epiphyseal dysplasia congenita (SEDC) family.Methods Five affected individuals from a Chinese SEDC family were enrolled in the study. All patients underwent thorough physical and radiographic examinations. DNA samples of the affected patients and the healthy controls were collected with the informed consent obtained from each participant. Two short tandem repeat (STR) polymorphic markers flanking COL2A1gene region were detected to determine the haplotype of each patient. Subsequently, sequence analysis was performed in COL2A1gene to identify potential genetic mutation.Results Haplotype analysis showed that the same disease-associated haplotype was segregated through the whole pedigree. A maximum LOD score of1.5was obtained with D12S85and D12S368. DNA sequence analysis revealed a c.1636G/A transition in exon25of the COL2A1gene, which converted the codon GGT for glycine at position546to AGT, a codon for serine. The patients were all heterozygous for the mutation G546S, which was absent in either of the unaffected family members or of the normal individuals.Conclusions This is the first familial report of G546S mutation in the COL2A1gene that results in SEDC. Although great achievements have been made in the recognition of the mutation spectrum, more intensive studies are warranted to further identify correlations between genotype and phenotype. |
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