osteoarthritis ( oa ) is a degenerative joint disease that progressively causes loss of joint function and is a major source of physical disability and impaired quality of life in many countries [ 13 ] .
the pathological changes which occur during oa involve all the joint structures , that is , synovium , cartilage , and bone tissues , but the main hallmark of this disease is the degradation of cartilage [ 4 , 5 ] .
aging , trauma , hormonal , and mechanical factors are reported to contribute to the onset and progression of oa [ 68 ] .
in addition , several studies have demonstrated the polymorphisms of some genes may be related to the pathogenesis of oa as well [ 911 ] .  
it is now accepted that the excessive and spontaneous inflammation plays a significant role in the molecular pathogenesis of oa , contributing to a highly catabolic state , chondrocyte apoptosis , and the resultant progressive degeneration of articular cartilage [ 1214 ] .
bradykinins , a family of oligopeptides derived from the enzymatic action of kallikreins on kininogens , can promote all the major signs of inflammation , including hyperemia , leakage of plasma proteins , and pain [ 1517 ] .
the presence of bk was previously reported in the synovial fluid from patients affected by arthritis of different etiologies , including oa [ 18 , 19 ] .
bdkrb2 has been detected on the synovial lining cells , fibroblasts , and endothelial lining cells of blood vessels from oa patients [ 18 , 21 ] .
clinically , the administration of b2 receptor antagonists effectively reduced the inflammatory articular pain and knee oa progression , suggesting the bdkrb2 is involved in the development of oa [ 18 , 22 ] .  
the genetic variants of bdkrb2 may lead to altered biological activities of the functional protein .
the gene polymorphisms of bdkrb2 have been shown to be related with acei - induced cough in hypertensive patients , left ventricular hypertrophy , and insulin resistance [ 2326 ] .
however , its relation with oa remains unknown . in this study , we enrolled knee oa patients to explore the role of bdkrb2 in oa .
a total of 245 patients with primary knee oa were recruited from dec 2008 to feb 2009 .
the severity of oa was evaluated according to the kellgren - lawrence ( kl ) grade classification , and only patients with k / l grades of 2 or higher were included .
both oa and control groups were interviewed to obtain demographic data and all of established risk factors . in the study , other etiologies causing knee diseases such as inflammatory arthritis ( rheumatoid , polyarthritic , or autoimmune disease ) , posttraumatic or postseptic arthritis , skeletal dysplasia , or developmental dysplasia were excluded from oa group .
all the control never had any signs or symptoms of arthritis or joint diseases ( pain , swelling , tenderness , or restriction of movement ) .
the clinical characteristics of all enrolled subjects , including age , sex , body mass index ( bmi ) , smoke status , bone fracture history , knee activity , and regular excise , were recorded .
the study was approved by the ethics review committee of our hospital , and written informed consent was obtained from all participants . reaction conditions for genotyping the two polymorphic loci ( + 9/9 and c 58 t ) were as follows : dna ( 100  ng ) was amplified in a 25  l reaction buffer containing 0.2  mmol / l deoxynucleotide triphosphates , 1.0  mmol / l mgcl2 , 20  mmol / l tris / cl ( ph = 8.4 ) , 50  mmol / l kcl , 0.015  nmol of each primer , and 0.5 u taq polymerase ( invitrogen corporation , carlsbad , ca , usa ) for 40 cycles of one minute at 94c , 30 s at 60c ( + 9/9 ) or 57c ( c 58 t ) , and 10  s at 72c , followed by a five - minute soak at 72c in a g - storm thermal cycler ( alphametrix biotech gmbh , rdermark , germany ) .
the primers for the bdkrb2 + 9/9 polymorphism were as follows : forward , 5-tccagctctggcttctgg-3 , and reverse , 5-agtcgctccctggtactgc-3 , and the amplification products were 80  bp ( 9 ) versus 89  bp ( + 9 ) .
the bdkrb2 c 58 t polymorphism was assayed using a pair of degenerate primers which were as follows : forward , 5-aaggtggccgcagccttcc-3 , and reverse , 5-ctcatctttcaagggctggcta-3. the reverse primer contained a g - c transversion ( underlined ) that generated a recognition site ( 5-ctag-3 ) for the restriction endonuclease bfai ( new england biolabs , ipswich , ma , usa ) in the presence of the c allele . if the c allele was present , the 133  bp pcr product digested to 112 + 21  bp .
all pcr or digestion products were size - separated by electrophoresis in 8% polyacrylamide gel electrophoresis gels run in 1  trisborate - edta buffer and visualized with ethidium bromide and ultraviolet light .
when observed or expected values included a cell with a value less than 5 , fisher 's exact test was used . in all cases ,
 
  or fisher tests were used to compare genotype frequency and demographic distributions between cases and controls .
multiple logistic regression analyses were used to evaluate if each snp was independently associated with oa when adjusted for the potential confounding effects of important clinical variables .
the odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated .
all analyses were performed by using spss software ( statistical package for the social sciences , version 16.0 , spss inc , chicago , il , usa ) .

 table 1 shows demographic and clinical characteristics of all subjects in the study . there were no significant differences in sex , age , smoke status , and history of heavy labor work between knee oa cases and controls .
obesity prevalence was significantly higher in the oa patient group than in controls ( p = 0.013 ) .  

table 2 described the genotype distributions and allele frequencies of bdkrb2 polymorphisms in knee oa and control subjects .
the genotype frequencies for all polymorphisms did not differ significantly from those expected under hardy - weinberg equilibrium ( both p > 0.05 ) .
the genotype frequencies and allele frequencies at bdkrb2 58t / c were similar between oa and control subjects .
the 9/9 genotype was significantly higher in knee oa subjects than in controls ( 33.46% versus 20.07% ) .
accordingly , the 9 allele frequency was higher in oa patients than controls ( 57.754% versus 47.34% , p < 0.001 ) .
logistic regression analysis showed a significantly increased risk for knee oa for the 9/9 genotype compared with the + 9/+9 genotype ( or = 2.35 , 95% ci : 1.4093.937 ; p < 0.001 ) after adjustment with sex , age , bmi , smoke status , history of labor work , regular exercise , and knee activity .
the adjusted or for 9 allele carriage was significantly higher than 9 allele carriage ( or = 1.52 , 95% ci : 1.1861.947 , p < 0.001 ) .  
all the oa patients were grouped into two subgroups : subjects with kl  3 and those with kl score > 3 ( table 3 ) .
we found that the + 9/+9 genotype was higher in those with kl score > 3 than in those with kl score < 3 .
the + 9/9 and 9/9 genotypes represented higher risks of being severer oa ( or = 3.09 , p < 0.001 , and or = 2.98 , p = 0.002 , resp . ) .
osteoarthritis ( oa ) is a painful and degenerating progressive disease of the joints which affects millions of patients worldwide . in this study
, we investigated whether bdkrb2 gene polymorphisms influence the susceptibility of oa in a chinese cohort .
our results showed that the 9/9 carriers had markedly higher risk for oa compared with + 9/+9 carriers .
besides , the + 9/9 and 9/9 genotypes represented higher risks of being severer oa than + 9/+9 carriers . to the best of our knowledge ,
this is the first study regarding the role of bdkrb2 gene polymorphisms in oa .  
it contributes to the initiation and maintenance of inflammation , to exciting and sensitizing sensory nerve fibres , thus producing pain , and to activating synoviocytes and chondrocytes which are the main cells involved in the homeostasis of synovial fluid and cartilage , respectively [ 29 , 30 ] .
the bdkrb2 is constitutively expressed in most tissues and is considered a stronger mediator of vasodilation and inflammation through increased production and release of nitric oxide [ 31 , 32 ] .   in humans
the bdkrb2 gene contains a number of polymorphic loci , including a nine - base insertion / deletion in the first exon of the gene ( + 9/9 , rs5810761 ) and c to t transition in the promoter region ( c 58 t , rs1799722 ) .
the 9  bp deletion ( 9 ) in the gene encoding the bdkrb2 is associated with expression of higher concentrations of receptor mrna , suggesting its strong functional relevance .
the + 9/9 genetic polymorphisms have been reported to be associated with a series of pathological conditions including coronary artery disease , systemic hypertension , and increased left ventricular mass associated with hypertension and pulmonary artery pressure [ 3638 ] . to our surprise , although the role of bdkrb2 in inflammation has been documented , we did not find any reports with regard to the genetic polymorphisms of bdkrb2 gene and inflammation . in this study , we firstly reported the role of genetic polymorphisms of bdkrb2 in oa
. we found that the 9  p polymorphisms , rather than the 58t / c polymorphisms , are not only associated with the oa risk but also the oa severity .
this finding suggests that the bdkrb2 + 9/9 polymorphisms may be used as a genetic marker for the onset and development of oa .
however , it should be pointed out that our study is preliminary , and the results need to be further confirmed in larger - scale study , ideally , in different ethnic populations .
 
 background . the b2-bradykinin receptor ( bdkrb2 ) has been reported to associate with onset and development of osteoarthritis ( oa ) ; however , the role of bdkrb2 genetic polymorphisms in oa remains unknown . 
 method . 
 a total of 245 patients with primary knee oa and 264 healthy volunteer were recruited . 
 bdkrb2 gene polymorphisms , 58t / c and + 9/9  bp polymorphisms , were genotyped . 
 results . 
 the genotype distributions and allele frequencies of + 9/9  bp polymorphisms significantly differed between oa and control subjects . 
 logistic regression analysis showed carriers with 9/9 genotype had a significantly increased risk for knee oa compared with the + 9/+9 genotype ( adjusted or = 2.356 , p < 0.001 ) . 
 the or for 9 allele carriage was significantly higher than + 9 allele carriage ( adjusted or = 1.52 , p < 0.001 ) . 
 the + 9/9  bp polymorphisms also determined the oa radiographic severity . 
 the presence of 9  bp was associated with severer oa . 
 the 58t / c polymorphisms did not affect oa risk and severity . conclusion . 
 the + 9/9  bp polymorphisms of bdkrb2 gene may be used as a genetic marker for the susceptibility and severity of oa . 