The Relationship Between The Polymorphism Of SREBP-2, IGFBP3 Gene And ANFH Risk.

Objective:Avascular necrosis of the femoral head (ANFH) is a complex disease caused by both genetic susceptibility and environmental risk factors. The studies of ANFH genes polymorphism may provide important scientific evidence for ANFH screening, early diagnosis and treatment. On the basic of research development in ANFH molecular pathogenesis, the SREBP-2, and IGFBP-3 genes were selected to as target genes. The integrated application of multiple innovative molecular techniques were used to investigate SREBP-2, and IGFBP-3 gene polymorphism, the relationship between polymorphisms and ANFH risk as well as clinical phenotype of ANFH,respectively. From molecular genetics and gene function, the screening of the key target molecules in relation to ANFH risk will be useful to elaborate the molecular targets of prevention from and treatment for ANFH.Methods:49 patients of ANFH were randomly selected from the ANFH cases treated in the clinical orthopedics, Japan Union Hospital of Jilin University from June,2009 to December.2010. On ANFH clinical diagnosis and staging, the ANFH patients were diagnosed and staged according to the Ficat diagnosis and staging criteria. And the pathological etiologies classification of the ANFH patients was such as idiopathic, alcohol-induced or steroid-induced situation.42 health control subjects were selected from healthy volunteers following the principle of informed consent and matching to the age, sex of ANFH group. Two single nucleotide polymorphisms (SNP) in the SREBP-2 gene, rs2267439 and rs2267443 and one single nucleotide polymorphisms (SNP) in the IGFBP3 gene, rs2453839 were selected from public databases and genotyped in 49 ANFH patients and 42 control subjects by using molecular sequencing genotyping. The significance of genes polymorphisms distribution, the relationship between genetic polymorphisms and clinical phenotype, and genes interactions in ANFH group and control group was finished by usingstatistical analysis software SPSS 10.0 etc.Results:The following results were obtained:1. Comprehensive application of molecular cloning, fluorescent quantitative PCR. molecular sequencing techniques.3 gene fragments of SREBP-2,IGFBP3 in cases group and control group were successfully cloned and the clear gene fragment bands and base sequence were confirmed by electrophoresis and sequencing techniques, respectively. And in this study, the gold standard sequencing methods, as the critical technological system, not only improved the specificity and sensitivity of the detection, but also provided the scientific basis for further the research of genetics susceptibility, the expression of transcriptional and translational levels of genes in ANFH.2. In the research system, it was confirmed by molecular sequencing technology that SREBP2 gene rs2267439SNP polymorphism showed CC, CT, and TT three haplotype and rs2267443SNP polymorphism was GG, AA and GA three haplotype. Though no statistical significance, rs2267439TT haplotype frequency and rs2267443 haplotype frequency in ANFH group showed increased tendency, compared to those of normal control group, suggesting that rs2267439TT haplotype and rs2267443 GA haplotype may increase the ANFH risk.3. Analysis to the interaction between two SNP revealed the frequency of both rs2267439 (T T) haplotype and rs2267443 (G/A) haplotype of SREBP-2 gene in ANFH group were significantly higher than those in control group, indicating the interaction between (T/T) and (G/A) haplotype may be associated with the increased ANFH risk. It also means the subjects carried two mutant haplotype, bothT/T and G/A, may have higher ANFH risk than those of carried single mutant haplotype.4. Gene sequencing results confirmed the three haplotype, CC, TT and CT of IGFBP3 gene rs2453839. Although the genotype and allele frequencies in ANFH group was no significant difference between ANFH and normal control group the TT haplotype frequency (52.0% vs 71.4%) and TC haplotype frequency (40.0% vs 20%)of ANFH group showed decreased and increased tendency, in relation to that of control group, respectively. The assessment between IGFBP3 gene rs2453839 polymorphism and the risk of ANFH remains to be investigated.5. Comparative analysis between the genotype and clinical manifestations demonstrated the course of disease in ANFH patients to carry TT genotype of IGFBP3rs2453839 was significantly shorter.compared to that of CT+CC-type carriers (P<0.01); in bilateralâ…¡â…¢stage patients, to carry TT genotype was more than CT+CC-type carriers (P<0.05); the TT genotype in the patients with unilateral hip involvement was also more than the TT-CT + CC-type carriers (P<0.05),suggesting the relation the between the gene polymorphism of IGFBP3rs2453839 and the clinical phenotype of ANFH.Conclusion:Our results firstly proposed the relevant evidence between the gene polymorphism of SREBP-2, IGFBP3 and ANFH risk in Northern Chinese, Han population. SREBP-2. and IGFBP3 polymorphism may be one of the most important genetic susceptibility factors for ANFH in Chinese Han population. The progress researches on the genetics of SREBP-2, and IGFBP3 genes, its epigenetics, gene expression efficiency, clinical phenotype may help to further elaborate gene polymorphisms with ANFH risk as well as propose the molecular level targets for the prevention and treatment of ANFH.