Significant association of SREBP-2genetic polymorphisms with avascular necrosis in the Korean population

Blood samples and information were obtained from 443 unrelated patients with AVN (366 men, 77 women; age: 49.7 ± 13.3) and 273 control subjects (206 men, 67 women; age: 52.1 ± 10.6) visiting Kyungpook National University Hospital (Daegu, Korea) between 2002 and 2006. Diagnosis was established by the evidence of symptomatic AVN using anteroposterior, lateral-pelvic radiographs and magnetic resonance imaging (MRI) in stage 1 of association research circulation osseous (ARCO) classification system, and by plain radiographs in stages 2, 3 and 4. Patients with a demonstrable history of direct trauma or with a possible combination of causes were excluded. Based on etiological factors, the patients were subgrouped into idiopathic (181 cases), alcohol-induced (206 cases) and steroid-induced (56 cases) groups. Steroid-induced AVN was defined by a history of taking 1800 mg prednisolone or an equivalent over 4 weeks with nephritic syndrome, systemic lupus erythematosus, rheumatoid arthritis, allergic asthma, or organ transplantation [15]. Alcohol-induced AVN was defined by the consumption of more than 400 ml of pure ethanol per week, or by the observation of alcohol induced fatty liver and liver cirrhosis. Control subjects were recruited from spouses of the patients and the general population and were considered normal if they had no hip pain and if anteroposterior and frog-leg lateral pelvic radiographs did not reveal any lesions with sclerotic margins or subchondral collapse consistent with AVN. All individuals provided informed consent for their participation in the study, and this project was approved by our Institutional Review Board.

Genomic DNA was isolated from peripheral blood leukocytes using a FlexiGene DNA Kit (QIAGEN, Valencia, CA, USA). All PCR reactions were performed in a 25 μl volume containing 1× PCR buffer (Solgent, Korea), 10 mM deoxynucleotide triphosphate (dNTP), 10 pmol each of forward and reverse oligonucleotide primer, 1 U/μl Taq DNA polymerase (Solgent, Korea), and 25 ng genomic DNA. The parameters for PCR consisted of a denaturation cycle at 95°C for 15 min, followed by 35 cycles at 95°C for 20 sec, 55–57°C for 40 sec, 72°C for 1 min, a final extension cycle at 72°C for 5 min, and a stabilization step at 4°C. PCR was performed using a PTC-200 thermal cycler and an iCycler™ Thermal cycler (BIO-RAD). For purification of the PCR products, an ExoSAP IT Kit (USB Corp., Cleveland, OH, USA) was used.

Genotyping was performed by the single-based extension (SBE) method with a SNaPshot Kit (Applied Biosystems Corp., Foster City, CA, USA). The SBE reaction mixture was prepared according to the manufacturer's instructions. The primer extension reaction was performed in 25 cycles at 96°C for 10 sec, 50°C for 5 sec, and 60°C for 30 sec. To resolve the excess primers, 1 unit of shrimp alkaline phosphatase (USB Corp., Cleveland, OH, USA) was added to the reaction mixture, and the mixture was incubated at 37°C for 60 min, followed by 15 min at 80°C for enzyme inactivation. The loading solution containing an injection marker was added to inactivate the SNaPshot reaction mixtures according to the recommendations of the manufacturer. Genotype scanning was performed using an ABI 3130 × l genetic analyzer, and the resulting files were analyzed by using Gene Mapper software program (Applied Biosystems Corp., Foster City, CA, USA).

The Hardy-Weinberg equilibrium (HWE) was tested to determine significant deviation of the genotype frequency from each single nucleotide polymorphism (SNP) by using the χ² test. Logistical regression analyses were used to calculate the odds ratios (ORs), 95% confidence intervals (CIs) and haplotypes, controlling for sex and age as covariates, with three alternative models (codominant, dominant and recessive). The linkage disequilibrium (LD) between loci was measured using the absolute value of Lewontin's D' (|D'|) and r ² [16]. Haplotypes of the SREBP-2 gene were analyzed using Haploview version 3.32http://​www.​broad.​mit.​edu/​haploview/​haploview based on the expectation maximization (EM) algorithm [17]. All analyses were two-tailed, and a P value < 0.05 was considered statistically significant. Statistics were performed using SAS 9.1 (SAS Institute Inc., Cary, NC, USA).