The Role And Function Of Susceptible Genes In AIS Based On Chinese Han Population

Chapter 2 The role of PIEZ02 gene in the proprioception of AIS Objective This study aims to investigate the proprioception of patients with AIS and the potential role of PIEZ02 gene in the development of AIS.Methods Proprioception tests were used to compare the difference in proprioception between AIS patients and aged matched healthy controls.Based on the falling risk scores(cut-off value was 36),AIS patients were divided into impaired proprioception group and unimpaired proprioception group.Paraspinal muscle was collected from 34 AIS patients during surgery.Student t test was used to compare the PIEZ02 gene expression between the impaired group and the unimpaired group.The average number of muscle fibers in the muscle spindle was compared between the AIS patients and controls.Results Proprioception test results showed that patients had significantly higher falling index and FK test value than the controls(41.7 ± 16.5 vs.11.3 ± 8.3,p=0.004;For distance of displacement,64.1 ± 16.3cm vs.42.2±7.9cm,p=0.003;For angle of rotation,25.20±20.1° vs 15.8°± 14.2°,p=0.002).In addition,the expression of PIEZ02 gene was remarkably decreased in the impaired group compared with the unimpaired group(0.51±0.24 vs.1.00±0.33,p=0.004).The average number of muscle fibers in the muscle spindle was significantly decreased in the AIS patients in the IP group than those in the UP group(2.2 ± 1.3 vs.3.5 ± 2.1,p=0.04).PIEZ02 expression level was positively correlated with the average number of muscle fibers in the muscle spindle(r=0.352,p=0.04).Conclusions Abnormal expression of PIEZ02 may play a role in AIS through impaired proprioception and a decreased number of muscle spindle in AIS.Further investigation is warranted to illustrate the role of PIEZ02 in the development of AIS.Chapter 3 Asymmetric expression of Wnt/β-catenin pathway in AIS:primary or secondary to the curve?Objective This study aims to investigate whether there exists asymmetric expression of Wnt/B-catenin pathway in the concave and the convex side of AIS and to clarify its relationship with the development of spinal deformity.Methods 3 groups of subjects were included in this study.Group 1 was composed of 40 female AIS patients aged between 10 and 18 years old.Group 2 was composed of 20 CS patients who were matched with AIS in terms of age,curve pattern and curve magnitude.Group 3 was composed of 24 adolescent female lumber disc herniation(LDH)patients with no spinal deformity.Paraspinal muscles were collected from all subjects during surgery.Specifically,for CS patients,the samples were collected from concave side and the convex side at the apical region.For AIS patients,the samples were collected from the proximal bilateral sides of the spine in addition to the apical region.For LDH patients,the samples were collected from the bilateral sides at L5 level.qPCR and western blot were used to determine the expression of 3 genes in Wnt/B-catenin pathway as previously reported,including LBX1,B-catenin and TNIK.Results The mean age were 14.4 ± 1.7 yrs for AIS,14.2±1.5 yrs for CS and 14.8 ±2.8 yrs for LDH,respectively.The mean Cobb angle was 49.1±10.7 degrees for AIS and 49.8 ± 11.2 degrees for CS,respectively.There were no difference in age among three groups.The mean Cobb angle was comparable between the AIS group and CS group.AIS patients were found to have remarkably lower mRNA and protein expression of B-catenin,LBX1 and PAX3 in the concave side than in the convex side at the apical region.By contrast,at the proximal region,the mRNA expression of these 3 genes were comparable.Moreover,no significant difference regarding mRNA expression was found between the concave side and the convex side of CS patients,or between the bilateral sides of LDH patients.Conclusions There exists remarkably asymmetric expression of Wnt/B-catenin pathway at the apex of AIS.The asymmetric expression of susceptible genes is likely to be primary to the curve,and may play a role in the etiology of AIS.Chapter 4(section 1)Investigating the role of IRX family in the development of female AIS:which one is the real cause?Objective This study aims to validate the relationship between IRX family and AIS in a large-scale general population and to further investigate the target gene of the region which was asscocaited with AIS.Methods SNP rs12517904 and rs117273909 was genotyped in 1323 patients and 1670 age-matched healthy controls.Paraspinal muscle was collected from 70 AIS patients and 20 congenital scoliosis patients.Student t test was used to compare the IRX expression between two groups.One-way ANOVA test was used to compare the expression of the IRX genes among different genotypes.The Pearson correlation analysis was carried out to investigate the relationship between the expression of the IRX1 gene and the curve severity.Results For rs12517904,patients were found to have a significantly higher frequency of TT than the controls(37.6%vs.34.7%,p=0.04).Allele T can significantly add to the risk of AIS with an OR of 1.14.AIS patients were found to have significantly lower IRX1 expression than the controls.Patients with genotype TT were found to have significantly lower IRX1 expression than those with genotype GGConclusions Our large-scale case-control study validated that the rs 12517904 of IRX1 gene is significantly associated with the development of AIS in Chinese population.Moeover,the decreased expression of IRX1 is significantly correlated with the curve severity of AIS.The functional role of IRX1 in the onset and progression of AIS is worthy of further investigation.Chapter 4(section 2)Rare variant of HSPG2 is not involved in the development of adolescent idiopathic scoliosis:evidence from a large-scale replication studyObjective This study aims to validate the relationship between rare variants of HSPG2 and AIS in a large-scale general population.Methods SNVs p.Asn786Ser of HSPG2 was genotyped in 1752 patients and 1584 normal controls using multiple ligase detection reactions.The mRNA expression of HSPG2 in the paraspinal muscles was quantified for 90 patients and 26 controls.The The Student’s t test was used to analyze the inter-group comparison of the HSPG2 expression.The relationship between the HSPG2 expression and the curve magnitude of the patients was analyzed by the Pearson correlation analysis.Results No case of mutation in the reported SNV p.Asn786Ser of HSPG2 was found in our cohort.The mRNA expression of HSPG2 in patients was comparable with that in the controls(0.0016 ± 0.0013 vs.0.0019 ± 0.0012,p=0.29).42 patients with curve magnitude>60 degrees were assigned to the severe curve group.The other 58 patients were assigned to the moderate curve group.These two groups were found to have comparable HSPG2 expression(0.0015±0.0011 vs.0.0017 ± 0.0014,p=0.57).And there was no remarkable correlation between the expression level of HSPG2 and the curve severity(r=0.131,p=0.71).Conclusions HSPG2 gene was not associated with the susceptibility or the phenotypes of AIS in the Chinese population.The whole HSPG2 gene can be sequenced in more AIS patients to identify potentially causative mutations.Chapter 4(section 3)Lack of association between AKAP2 and the susceptibility of adolescent idiopathic scoliosis in the Chinese population Objective To investigate whether genetic variants of AKAP2 are associated with the susceptibility of adolescent idiopathic scoliosis(AIS)in Chinese population.Methods SNV c.2645A>C of AKAP2 was genotyped in 1254 AIS patients and 1232 normal controls using allelic-specific multiple ligase detection reactions.SNPs located within 5’ untranslated regions(UTR)and 3’UTR of AKAP2 gene were selected using Haploview(v2.6).The GWAS database composed of 961 AIS patients and 1499 controls was referred to for the genotyping information.Relative mRNA expression of AKAP2 in peripheral blood was analyzed for 33 patients and 18 age-matched controls.Comparison between the cases and controls were performed using the Student’s t test.PLINK(vl.90)was used to calculate the association of each SNP with the disease by Cochran-Armitage trend test.Results All the patients and the controls presented a genotype of AA in c.2645A>C of AKAP2,and there was no case of mutation in any subject.A total of 116 SNPs covering AKAP2 were analyzed,and none of these SNPs was found to have significantly different allele frequency between the cases and the controls.The mRNA expression of AKAP2 in patients was comparable with that in the controls(1.9 ± 0.8 vs.1.8 ± 0.7,p=0.66).Conclusions Our large-scale replication study of the variants in AKAP2 gene did not support its association with the susceptibility of AIS in the Chinese population.In future study,functional studies of the previously reported rare variant is warranted to clarify whether the variant can regulate the expression of AKAP2.The whole AKAP2 gene can be sequenced in larger AIS cohorts to identify potentially missing mutations.