obesity is one of the most common disorders in clinical practice and closely associated with a number of pathological diseases , such as non - insulin - dependent diabetes , hypertension , and atherosclerosis ( gregoire et al . , 1998 ) . however , the greatest proportion of obesity involves the interaction variants in multiple genes with environmental factors , particularly diet ( loos & bouchard , 2003 ; loss & rankinen , 2005 ) . in addition , several lines of studies suggested a close association between insulin signaling and obesity . ( 2003 ) have reported the molecular mechanisms of insulin resistance and obesity , focusing on the function of adipocyte . pre - b - cell leukemia transcription factor ( pbx1 ) is a member of a ubiquitous class of homeodomain proteins . the pbx1 gene , localized on chromosome 1q23 , was originally identified by its involvement in a t ( 1;19 ) chromosomal translocation in acute pre - b - cell leukemias ( kamps , 1990 ; nourse et al . , subsequent study has shown that pbx1 is widely expressed and acts as a cofactor for a variety of hox proteins ( sagerstrom , 2004 ) . these homeodomain transcription factors in turn regulate developmental gene expression and early embryonic development and organogenesis , and maintain differentiated cell states ( sagerstrom , 2004 ) . the role of pbx1 has been well studied in the pancreas , where it complexes with and regulates the key -cell homeobox protein , pdx1 ( ipf1 in humans ) ( wang et al . , 2005 ) . in mice , pdx1 activity was sufficient for the development of the major cell types , but pbx1:pdx1 complex was essential for the expansion of each cell type within the developing pancreas , and hence for normal pancreatic development ( dutta et al . , 2001 ) . ( 2001 ) also suggested that pbx1:pdx1 heterodimer contributes to ductal cell proliferation and subsequent islet regeneration . 2002 ) examined pancreatic pbx1 expression , morphogenesis , cell differentiation , and function in mice deficient for pbx1 . embryos had pancreatic hypoplasia and marked defects in exocrine and endocrine cell differentiation prior to death at embryonic day 15 or 16 . 2002 ) concluded that pbx1 is essential for normal pancreatic development and function and perturbation of pbx1 activity may also promote susceptibility to diabetes mellitus . despite potentially important roles in metabolic diseases , genetic association of pbx1 on obesity therefore , we examined whether pbx1 polymorphism contributes to the susceptibility to obesity or obesity - related metabolism traits in a korean population . the body mass index ( bmi ) was calculated from height and weight using the formula : bmi = body weight/(height ) in kg / m . in the world health organization guidelines for asians , individuals with a bmi23 kg / m are classified as overweight and those with a bmi25 kg / m are defined as obese ( world health organization expert consultation , 2004 ) . we recruited a case group ( n=214 ) that comprised overweight and obese subjects ( bmi23 kg / m , 123 men and 91 women ) and control subjects ( n=160 ) with normal weight ( bmi : 18.0 - 22.9 kg / m , 66 men and 94 women ) at kyung hee university medical center and keimyung university dongsan medical center . blood samples were drawn for biochemical measurements , i.e. , fasting plasma glucose , hemoglobin a1c ( hba1c ) , triglyceride ( tg ) , total cholesterol , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) . dna was isolated from peripheral blood leukocytes using the g - dex iib genomic dna extraction kit ( intron biotechnology , seongnam , korea ) . this study was approved by the institutional review board of kyung hee university medical center . initially , we selected 66 single nucleotide polymorphisms ( snps ) within the pbx1 gene using the following criteria : ( 1 ) consisting only of snps in introns ( approximately 300bp from the exon to consider alternative splicing ) ; ( 2 ) tagging snps selected using the tagging option of the program tagger ( http://www.broad.mit.edu/mpg/tagger/ ) ; ( 3 ) known heterozygosity and minor allele frequency ( maf ) > 0.05 ; and ( 4 ) reported validation ( http://www.hapmap.org ) . the genotyping was performed using the affymetrix targeted genotyping chip array ( affymetrix , ca , usa ) , according to the manufacturer 's protocol . this chip uses molecular inversion probe technology with gene chip universal microarrays to provide a method that is capable of analyzing thousands of variants in a single reaction . in brief , dna was digested and then subjected to pcr using primers specific to the adaptor sequence . pcr products were purified and the fragmented dna was then end - labeled with biotin using terminal deoxynucleotidyl transferase . the hybridized array was washed , stained , and scanned according to the manufacturer 's instructions . for the case - control association study , hardy - weinberg equilibrium ( hwe ) for all snps was assessed using snpstats . ( sol et al . , 2006 ) a linkage disequilibrium ( ld ) block of polymorphisms was tested using haploview ( version 3.32 ) ( ma , usa ) . ( barrett et al . , 2005 ) multiple logistic regression models were calculated for the odds ratio ( or ) , 95% confidence interval ( ci ) and corresponding p values , controlling for age and gender as covariables . we used snpstats , hapanalyzer version 1.0 ( seoul , korea ) ( park et al . , 2008 ) and snpanalyzer ( istech inc . , the power of the sample size was calculated using a genetic power calculator ( http://pngu.mgh.harvard.edu/~purcell/gpc ) ( purcell et al . , 2003 ) . to reduce experimental error , the effective sample size was adjusted ( calculated sample size100/95 ) . we calculated sample powers for the snps to confirm the effects . to account for multiple testing , the bonferroni method was used ( bland & altman , 1995 ) , giving a significance level of /=0.00076 for each test . clinical characteristics were compared between control and case subjects , using student 's unpaired t - test for continuous variables with normal distribution including systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , fasting plasma glucose , hba1c , tg , total cholesterol , hdl - c , and ldl - c . in addition , the statistical significance of association between the genetic variants and clinical characteristics in both the control and case groups was assessed using one - way analysis of variance ( anova ) with subsequent tukey 's tests . for all of the statistical tests , the body mass index ( bmi ) was calculated from height and weight using the formula : bmi = body weight/(height ) in kg / m . in the world health organization guidelines for asians , individuals with a bmi23 kg / m are classified as overweight and those with a bmi25 kg / m are defined as obese ( world health organization expert consultation , 2004 ) . we recruited a case group ( n=214 ) that comprised overweight and obese subjects ( bmi23 kg / m , 123 men and 91 women ) and control subjects ( n=160 ) with normal weight ( bmi : 18.0 - 22.9 kg / m , 66 men and 94 women ) at kyung hee university medical center and keimyung university dongsan medical center . blood samples were drawn for biochemical measurements , i.e. , fasting plasma glucose , hemoglobin a1c ( hba1c ) , triglyceride ( tg ) , total cholesterol , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) . dna was isolated from peripheral blood leukocytes using the g - dex iib genomic dna extraction kit ( intron biotechnology , seongnam , korea ) . this study was approved by the institutional review board of kyung hee university medical center . initially , we selected 66 single nucleotide polymorphisms ( snps ) within the pbx1 gene using the following criteria : ( 1 ) consisting only of snps in introns ( approximately 300bp from the exon to consider alternative splicing ) ; ( 2 ) tagging snps selected using the tagging option of the program tagger ( http://www.broad.mit.edu/mpg/tagger/ ) ; ( 3 ) known heterozygosity and minor allele frequency ( maf ) > 0.05 ; and ( 4 ) reported validation ( http://www.hapmap.org ) . the genotyping was performed using the affymetrix targeted genotyping chip array ( affymetrix , ca , usa ) , according to the manufacturer 's protocol . this chip uses molecular inversion probe technology with gene chip universal microarrays to provide a method that is capable of analyzing thousands of variants in a single reaction . in brief , dna was digested and then subjected to pcr using primers specific to the adaptor sequence . pcr products were purified and the fragmented dna was then end - labeled with biotin using terminal deoxynucleotidyl transferase . the hybridized array was washed , stained , and scanned according to the manufacturer 's instructions . for the case - control association study , hardy - weinberg equilibrium ( hwe ) for all snps was assessed using snpstats . ( sol et al . , 2006 ) a linkage disequilibrium ( ld ) block of polymorphisms was tested using haploview ( version 3.32 ) ( ma , usa ) . ( barrett et al . , 2005 ) multiple logistic regression models were calculated for the odds ratio ( or ) , 95% confidence interval ( ci ) and corresponding p values , controlling for age and gender as covariables . we used snpstats , hapanalyzer version 1.0 ( seoul , korea ) ( park et al . , 2008 ) and snpanalyzer ( istech inc . , the power of the sample size was calculated using a genetic power calculator ( http://pngu.mgh.harvard.edu/~purcell/gpc ) ( purcell et al . , 2003 ) . to reduce experimental error , the effective sample size was adjusted ( calculated sample size100/95 ) . we calculated sample powers for the snps to confirm the effects . to account for multiple testing , the bonferroni method was used ( bland & altman , 1995 ) , giving a significance level of /=0.00076 for each test . clinical characteristics were compared between control and case subjects , using student 's unpaired t - test for continuous variables with normal distribution including systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , fasting plasma glucose , hba1c , tg , total cholesterol , hdl - c , and ldl - c . in addition , the statistical significance of association between the genetic variants and clinical characteristics in both the control and case groups was assessed using one - way analysis of variance ( anova ) with subsequent tukey 's tests . for all of the statistical tests , all of the measured clinical characteristics differed significantly between the case and control groups ( table 1 ) . we analyzed the association between each genotype and susceptibility to obesity using logistic regression analysis , after adjusting for age and gender . the genotype distributions of all snps selected were in hardy - weinberg equilibrium ( p>0.05 ) . of these , seven snps ( g.+75186c > t , g.+78350c > a , g.+80646c > t , g.+138004c > t . g.+185219g > a , g.+191272a > c , and g.+265317t > a ) were significantly associated with the risk of obesity ( p<0.05 ; table 2 ) . t , g.+185219g > a , and g.+191272a > c had a protective effect in the dominant ( p=0.029 , or=1.68 , 95% ci=1.05 - 2.69 ) , recessive ( p=0.05 , or=1.75 , 95% ci=1.00 - 3.05 ) , codominant ( p=0.034 , or=1.51 , 95% ci=1.03 - 2.21 ) , codominant ( p=0.035 , or=1.68 , 95 % ci=1.04 - 2.72 ) models , respectively . conversely , the minor allele of g.+78350c > a and g.+265317t > a showed susceptibility to the risk of obesity when a recessive model was applied ( p=0.007 , or=0.46 , 95% ci=0.25 - 0.83 , and p=0.02 , or=0.14 , 95% ci=0.02 - 1.12 , respectively ) . in addition , g.+80646c > t was statistically associated with obesity in all models . however , this association disappeared after correcting for multiple testing by the bonferroni test . ( gabriel et al . , 2002 ) of these blocks , six ld blocks containing seven significant snps were used for further haplotype association analysis . haplotypes 1 ( cac ) and 3 ( tac ) of block 3 , which consisted of g.+80646c > t , g.+87440a > g , and g.+91850c > t , showed a significant association with obesity in the dominant ( p=0.007 , or=0.45 , 95% ci=0.26 - 0.81 ) and codominant ( p=0.027 , or=1.54 , 95% ci=1.05 - 2.25 ) models , respectively ( table 3 ) . haplotype 2 ( ggaat ) of block 10 , which comprised of g.+185286g > a , g.+185602g > a , g.+191125a > g , g.+191272a > c , and g.+194899g > t , was also significantly associated with the risk of obesity in the codominant ( p=0.029 , or=1.59 , 95% ci=1.05 - 2.42 ) and recessive ( p=0.034 , or=1.64 , 95% ci=1.04 - 2.59 ) models . however , when bonferroni 's correction for the multiple comparisons was strictly applied , no haplotype also remained significant in terms of the association with obesity . for the association analysis of obesity - related phenotypes , we assessed whether the obesity - related phenotypes differed between the two groups ( case and control ) according to the pbx1 polymorphisms . there were significant differences in sbp , dbp , tg , total cholesterol , hdl - c , and ldl - c , except fasting plasma glucose and hba1c , between the control and case group for the g.+185219g > a and g.+191272a > c ( table 4 ) . in the control group , subjects that carried the minor allele had higher hdl - cholesterol level compared to the homozygote for the major allele for both g.+185219g > a and g.+191272a > c ( 59.8  14.9 vs 54.3  10.9 and 61.3  15.3 vs 55.3  11.9 , respectively , p<0.001 ) . as shown in table 1 , hdl - cholesterol level was significantly lower in the case group compared to the control group ( 49.8  11.3 vs 56.9  13.1 , p<0.001 ) . therefore , these results suggest that the association between the homozygote for the major allele for both g.+185219g > a and g.+191272a > c and hdl - c was attributable to an increased risk of obesity . obesity is a common complex disease that involves multiple genetic variants interacting with environmental and behavioral factors , and is associated with an increased risk of developing insulin resistance and type 2 diabetes . pbx1 gene , comprised of nine exons spanning over 285 kb , maps to a region of chromosome 1 that shows well - replicated linkage to type 2 diabetes in multiple ethnic groups . several studies support an association between pbx1 polymorphisms and type 2 diabetes mellitus ( elbein et al . , 1999 ; mccarthy , 2003 ; ng et al . , 2004 ; thameem et al . , we evaluated the gene as a possible candidate gene for obesity based on the close relationship between obesity and type 2 diabetes . our results suggest that some snps in the pbx1 gene contribute to obesity and metabolic alterations in a korean population . in a case - control analysis , of the 66 snps examined in pbx1 , seven snps ( g.+75186c > t , g.+78350c > a , g.+80646c > t , g.+138004c > t , g.+185219g > a , g.+191272a > c , and g.+265317t > a ) were significantly associated with the risk of obesity . moreover , haplotypes 1 ( cac ) and 3 ( tac ) of block 3 , consisted of g.+80646c > t , g.+87440a > g , and g.+91850c > t , and haplotype 2 ( ggaat ) of block 10 , comprised of g.+185286g > a , g.+185602g > a , g.+191125a > g , g.+191272a > c , and g.+194899g > t , of the pbx1 gene had a significant association with the risk of obesity . to our best knowledge , this is the first demonstration that pbx1 polymorphisms are associated with susceptibility to obesity . interestingly , a strong association was also found between some snps and the hdl - c level in the control group . control subjects that possessed minor allele g.+185219g > a and g.+191272a > c had higher hdl - c levels . therefore , the minor allele g.+185219g > a and g.+191272a > c may decrease the risk of obesity . recently , although there were many reports about individual gene polymorphisms on hdl - c levels ( chien et al . , to our knowledge , this is the first report on the association between pbx1 polymorphisms and hdl - c levels . first , many gene variants contribute to obesity and related alterations and those in pbx1 may have modest effects , requiring large sample sizes to detect associations ( hirschhorn & daly , 2005 ) . our sample size was small , and it is possible that the associations that we observed between pbx1 snps and obesity could represent false positives . in our study , the power of the sample size was calculated using a genetic power calculator . our sample provides the power more than 80% to detect more than 1.7-fold increased risk assuming an -level of 0.05 . thus , our case - control study was sufficiently powerful to determine a positive association . second , all snps selected in pbx1 are located in introns and do not cause an amino acid change . according to the dssnp database ( http://www.ncbi.nlm.nih.gov/snp/ ) and the hapmap database ( http://www.hapmap.org/ ) , five snps in exons of pbx1 gene were reported , but there are unknowns about heterozygosity of them . for this reason we suggest that significant polymorphisms in introns may be a marker rather than a direct contributor to the genetic functions . however , we can not exclude a possibility that these intronic polymorphisms have a role on its genetic function via the change of alternative splicing ( ast , 2004 ) . in conclusion , our findings suggest that polymorphisms in pbx1 gene might be one of the genetic factors for the risk of obesity , especially in association with alteration of hdl - c level , in a korean population . further studies are needed to explore the specific association between g.+185219g > a and g.+191272a > and hdl - c .
pre - b - cell leukemia transcription factor 1 ( pbx1 ) , which is located on chromosome 1q23 , was recently reported to be associated with type 2 diabetes mellitus . 
 we examined whether single nucleotide polymorphisms ( snps ) of the pbx1 gene are associated with overweight / obesity in a korean population . 
 we genotyped 66 snps in the pbx1 gene and investigated their association with clinical phenotypes found in 214 overweight / obese subjects and 160 control subjects using the affymetrix targeted genotyping chip array . 
 seven snps ( g.+75186c > t , g.+78350c > a , g.+80646c > t , g.+138004c > t , g.+185219g > a , g.+191272a > c , and g.+265317t > a ) were associated with the risk of obesity in three models ( codominant , dominant , and recessive ) ( p=0.007 - 0.05 ) . 
 haplotype 1 ( cac ) and 3 ( tac ) of block 3 and haplotype 2 ( ggaat ) of block 10 were also strongly associated with the risk of obesity . in the control group , 
 subjects that had homozygote for the major allele for both g.+185219g > a and g.+191272a > c showed lower high density lipoprotein - cholesterol ( hdl - c ) level compared to those possessing the minor allele , suggesting that the association between the homozygote for the major allele for both g.+185219g > a and g.+191272a > c and hdl - c is attributable to the increased risk of obesity . 
 this study suggests that the pbx1 gene is a possible risk factor in overweight / obese patients .