BACKGROUND
metabolic syndrome, one of the main global challenges for modern health care  <cit> , involves elevated risk of several interrelated disorders: obesity, ischemic heart disease, hypertension, insulin resistance, type ii diabetes mellitus, and dyslipidemia.
it is commonly believed that obesity is among the key risk factors of metabolic syndrome  <cit> .
obesity affects quality of life and life expectancy and is associated with cardiovascular disorders  <cit>  , cancer  <cit> , diabetes  <cit> , and damage to kidneys and liver  <cit> .
it has also been reported that obesity correlates with reproductive disorders in women  <cit> , prostate diseases in men  <cit> , and elevated risk of congenital anomalies in children  <cit> .
the discovery  <cit>  that the leptin gene lep is "the obesity gene"  shed light on the regulation of energy metabolism  <cit> .
leptin is a peptide hormone with molecular mass ~ <dig> kd  <cit> .
its secretion by adipocytes is directly proportional to adipose-tissue weight.
recessive mutations in the leptin gene are strongly associated with obesity in mice and humans  <cit> .
leptin circulates with blood and regulates food consumption and energy demand of the brain.
with a decrease in fat weight, the plasma leptin level decreases to increase appetite and suppress energy consumption until the recovery of fat weight.
when fat weight increases, the level of circulating leptin increases and suppresses appetite until a fat weight reduction  <cit> .
initially, it was believed that leptin is produced mainly by adipocytes  <cit> , but later it was found that leptin is also produced in such organs as the stomach  <cit> , heart  <cit> , and placenta  <cit> .
in addition to body weight control, leptin plays other physiological roles.
it modulates vascular tone and blood pressure and enhances angiogenesis and calcification of vascular cells  <cit> .
numerous studies showed a direct correlation between blood leptin concentrations and blood pressure  <cit> .
elevated blood pressure contributes to atherosclerosis  <cit> .
recent studies on the development of obesity in male wistar rats uncovered an association of diet-induced fatty liver  with insulin/leptin resistance  <cit> .
recent years have witnessed an increasing number of women with metabolic imbalance during pregnancy, with a variety of consequences; the mechanism of this metabolic imbalance is still poorly understood  <cit> .
the molecular pathways linking obesity to the above disorders are unclear  <cit>  mostly because of the large number of genes related to the nervous system, endocrine system, and metabolic system that regulate energy metabolism.
the contribution of a single gene is estimated to be 1-6%  <cit> , which is close to the statistical significance threshold with the current accuracy of these estimates.
postgenomic predictive preventive medicine  <cit>  offers a way around this obstacle  by taking into account numerous snp markers of various partially obesity-related complications in the above-mentioned or other disorders  <cit> .
analysis of snp markers, which show differences between an individual human genome and the reference human genome , as part of postgenomic preventive personalized medicine allows for effective treatment  <cit> , improvement of treatment  <cit> , and prevention of complications of treatment  <cit> .
genome-wide snp identification is the goal of the project  <dig> genomes  <cit> .
the dbsnp database  <cit>  documents current results of this project  <cit> ; thus, the reference human genome, hg <dig>  which is thought to contain the ancestral versions of all snps, is constantly refined.
it is available in the ensembl database  <cit>  via the web service ucsc genome browser  <cit> .
ensembl contains data on gene knockouts in animals, and this information is helpful, for example, for reconstruction of perturbation networks of various disorders and for development of therapeutic strategies  <cit> .
computer-based analysis of hundreds of millions of unannotated snps in  <dig> genomes  <cit>  that are documented in the dbsnp database  <cit>  may accelerate the search for biomedical snp markers  <cit> .
for this purpose, all the identified snps were mapped onto whole-genome maps of genes  <cit>  and onto protein-binding sites in dna that were predicted in silico  <cit>  and detected in vivo using chromatin immunoprecipitation , interchromosomal contacts, nucleosomes, transcriptomes either in health  <cit> , during infection  <cit> , disease  <cit> , or after treatment  <cit> .
on the basis of these data, many web services such as tfbs  <cit> , activity  <cit> , is-rsnp  <cit> , regulomedb  <cit> , rsnp-mapper  <cit> , raven  <cit> , selex_db  <cit> , funseq <dig>  <cit> , apeg  <cit> , featurescan  <cit> , snpchiptools  <cit> , snp-med  <cit> , snap  <cit> , funcisnp  <cit> , spot  <cit> , rsnp_guide  <cit> , and chromos  <cit>  facilitate the search for candidate snp markers in terms of ranking of unannotated snps by their similarity to biomedical snp markers in accordance with projections of these snps onto whole-genome maps.
according to the central limit theorem, the accuracy of this similarity-based search for candidate snp markers should increase as the number, diversity, representativeness, and completeness of genome-wide maps increase  <cit> .
due to this mainstream approach, the most impressive progress has been achieved with snps located in protein-coding gene regions  <cit>  because of the invariant types of disruption in both structure and function of the altered proteins regardless of the cellular conditions  <cit> .
on the other hand, the effects of snps that are located in regulatory regions of genes  <cit>  are still difficult to predict in silico  <cit> .
most studies in this field deal with regulatory snps in binding sites of tata-binding protein  in the region  upstream of the transcription start of various mrnas encoded in the human genome  <cit>  .
for this reason, model animals with a null-mutation  <cit>  or a knockdown of tbp  <cit>  are always inviable.
previously, we developed a computer-assisted method for estimation of significance  of the difference between ancestral and minor snp variants in terms of their effects on gene expression  <cit> .
later, we confirmed the predictions of this method by our independent experiments in vitro under both equilibrium  <cit>  and nonequilibrium  <cit>  conditions of the electrophoretic mobility shift assay .
then, we verified these empirical findings using a wide range of real-time assays, such as surface plasmon resonance, stopped-flow   <cit> , and fluorescence resonance energy transfer   <cit> .
in addition, we utilized independent results of over  <dig> experiments by others  <cit> .
on the basis of this comprehensive validation  <cit>  of the method  <cit> , we designed the web service snp_tata_comparator   <cit>  for the researchers who would like to analyze certain changes in core promoters of human genes.
in the present work, we cross-validated the output of two computer-based methods: dna sequence analysis using web service snp_tata_comparator and keyword search for articles on biochemical markers of obesity.
near the tbp-binding sites of human gene promoters, we found  <dig> obesity-related candidate snp markers, including rs <dig> , rs <dig> and rs <dig>  in the human gene  of leptin and rs <dig>  in the human gene  of glucagon.
we characterized them empirically and in terms of quantitative estimates of the equilibrium dissociation constant .
we then validated these estimates in terms of the apparent dissociation constant, k*d ≡ kd/ka, the ratio of the association rate constant , and the dissociation rate constant  of the tbp-dna complex by means of emsa under nonequilibrium conditions in vitro.
we found a significant linear correlation  between k0d values predicted in silico and k*d values determined by measurements in vitro for the obesity-related candidate snp markers rs <dig>  rs <dig>  rs <dig>  and rs <dig>  which may be useful for postgenomic predictive preventive personalized medicine.
RESULTS
the results of in silico analysis of the known and candidate snp markers in the tbp-binding sites of the human gene promoters
we applied our web service snp_tata_comparator  <cit>  to  <dig> biomedical and candidate snp markers in the tbp-binding sites of the human gene promoters taken from our recent review  <cit> .
table s <dig>  shows the results.
the human il1b gene
promoter contains the biomedical snp marker rs <dig>  this snp is associated with greater body fat in older men  <cit> , graves' disease  <cit> , gastric cancer  <cit> , hepatocellular carcinoma  <cit> , non-small cell lung cancer  <cit> , gastric ulcer and chronic gastritis  <cit> , and major recurrent depression .
it is a widely studied regulatory snp marker in the tbp-binding sites of human gene promoters.
previously, we also studied this snp in vitro by emsa under both equilibrium  <cit>  and nonequilibrium  <cit>  conditions as well as in silico  <cit> .
the human apoa <dig> gene
has the -35a→c substitution relative to the start of transcript number  <dig> of this gene.
this snp is inside a proven tata box   <cit> .
it is associated with obesity, fatty liver, and hematuria  <cit> .
using this snp marker of obesity, we validated the suitability of our web service snp_tata_comparator for analysis of obesity-related polygenic human diseases .
the human nos <dig> gene
contains an snp marker of epilepsy  <cit>  and resistance to malaria  <cit> .
it is a -51t→c substitution relative to the start of transcript number  <dig>  <cit>  that causes nos <dig> overexpression according to clinical research  <cit> .
we have previously studied this snp in depth using emsa  <cit>  and our computer-based method  <cit> .
in this work, an additional keyword search  pointed to new data suggesting that nos <dig> overexpression may be a biochemical marker of obesity  <cit> .
on the basis of this empirical observation  <cit> , we propose the -51t→c substitution in the nos <dig> gene promoter as a candidate snp marker of obesity .
the human pgr gene
contains the biomedical snp marker rs <dig> causing de novo appearance of a spurious tbp-biding site along with an additional pathogenic transcription start site  at position + <dig> relative to the normal tss for transcript number  <dig> of the same gene.
this snp is associated with endometrial cancer in obese women  <cit> .
it was previously analyzed by our computer-based method  <cit> .
here, using a keyword search, we found new clinical data showing that pgr overexpression is a biochemical marker of male breast cancer  in obesity  <cit> .
with this in mind, we propose rs <dig> as a candidate snp marker of breast cancer in obese men .
the human esr <dig> gene
contains the snp marker  of a primary esr2-deficient pt <dig> tumor that can transform into breast cancer without preventive treatment  <cit> .
an additional keyword search pointed to new clinical data showing that an esr <dig> deficiency can reduce the risk of obesity after ovariectomy  <cit> .
according to these data, we propose the snp s <dig> as a candidate marker of the low risk of obesity after ovariectomy .
the human hsd17b <dig> gene
contains an snp marker  of breast cancer  <cit> .
nevertheless, using a keyword search, we found a new promising study suggesting that weight loss in hsd17b1-deficient obese postmenopausal women by means of the appropriate diet and/or exercise reduces the risk of obesity-related cancers  <cit> .
thus, we propose rs <dig> as a possible predictive snp marker of effectiveness of preventive anticancer treatment among obese postmenopausal women .
the human mbl <dig> gene
contains the snp marker  of variable immunodeficiency  <cit> , preeclampsia  <cit> , and stroke  <cit>  caused by deficient expression of this gene as we have also predicted in silico  <cit>  and confirmed under both equilibrium  <cit>  and nonequilibrium  <cit>  conditions in vitro.
a keyword search for "mbl <dig> " "deficiency," and "obesity" pointed to clinical findings that an mbl <dig> deficiency can increase the risk of obesity  <cit> .
therefore, rs <dig> may be a candidate snp marker of obesity .
near this biomedical snp marker, we found one more snp: rs <dig> , and then, predicted that it can impair the expression of mbl <dig>  too.
this is why we propose rs <dig> as a candidate snp marker of obesity  <cit>  .
the human f <dig> gene
: its promoter contains a biomedical snp, namely, the -35a→c substitution relative to the start of transcript number  <dig> of this gene.
this snp is a marker of moderate bleeding caused by an f <dig> deficiency  <cit> .
an additional keyword search pinpointed the clinical finding of a statistically significant correlation between the total f <dig> level in obese patients with type  <dig> diabetes mellitus and the risk of cardiovascular complications  <cit> .
hence, we suggest the -35a→c substitution within the human f <dig> gene promoter as a candidate snp marker of the low risk of cardiovascular complications in obese patients with type  <dig> diabetes mellitus .
near this biomedical snp marker, we found two unannotated snps, rs <dig> and rs <dig>  that can cause overexpression of the f <dig> gene .
on the basis of the correlation  <cit> , we propose the two snps rs <dig> and rs <dig> as candidate markers of the high risk of cardiovascular complications in obese patients with type  <dig> diabetes mellitus .
the human f <dig> gene
contains a known snp marker  of obesity  <cit> , myocardial infarction, and thromboembolism caused by f <dig> overexpression  <cit>  .
the human hbb and hbd genes
contain the best-studied tbp-binding sites that are altered by a number of snp markers of resistance to malaria and thalassemia   <cit> , namely: rs <dig>  rs <dig>  rs <dig>  rs <dig>  rs <dig>  rs <dig>  and rs <dig>  previously, we have analyzed most of them in depth using our computer-based method  <cit>  as well as using emsa under both equilibrium  <cit>  and nonequilibrium  <cit>  conditions in vitro.
using a keyword search, we found new clinical data showing that a hemoglobin deficiency can serve as a biochemical marker of chronic inflammation in comorbidities of obesity.
this finding allows us to propose rs <dig>  rs <dig>  rs <dig>  rs <dig>  rs <dig>  rs <dig>  and rs <dig> as candidate snp markers of inflammatory complications in obesity .
near these biomedical snp markers, we found three unannotated snps: rs <dig>  rs <dig>  and rs <dig>  our analysis predicted that they can cause a hemoglobin deficiency.
consequently, we propose them as candidate snp markers of inflammatory complications of obesity .
after that, we analyzed all unannotated snps in the  region  in the only known promoter of the human lep gene, which we selected due to friedman's discovery that the lep gene  is the "obesity gene": ob ≡ lep  <cit>  .
table s <dig>  shows three unannotated snps--rs <dig>  rs <dig>  and rs34104384--which can alter tbp's affinity for this promoter according to our prediction .
the first one in the list can reduce the tbp-promoter affinity, whereas the two others can increase this affinity.
using a keyword search, we found literature data showing that the leptin deficiency is a biochemical marker of obesity  <cit> , whereas leptin overexpression can serve as a marker of obesity-caused hypertension  <cit> .
this is why we propose three more candidate snp markers  of obesity-caused hypertension .
finally, we selected one more unannotated snp  within the tbp-binding site of the promoter of the human gcg gene of glucagon because this snp's association with obesity has not yet been examined.
similarly, we predicted a gcg deficiency and found  that it is a biochemical marker of the resistance to obesity during a high-fat diet  <cit> .
accordingly, we propose rs <dig> as a candidate snp marker of obesity resistance during a high-fat diet .
we reviewed all of the above findings and selected four of our obesity-related candidate snp markers--rs <dig>  rs <dig>  rs <dig>  and rs183433761--for empirical biochemical verification using emsa under nonequilibrium conditions in vitro because these snps have not yet been verified experimentally on their possible association with obesity.
the results of in vitro analysis of the four selected candidate snp markers
the primary experimental data from the in vitro analysis of the four selected candidate snp markers are depicted in figure  <dig>  table  <dig> shows these results for rs <dig>  rs <dig>  rs <dig>  and rs <dig> in terms of the primary experimental ka and kd values that characterize the kinetics of tbp's binding to the ancestral and minor variants of the tbp-binding site in the only promoter of the lep gene that is documented in the human reference genome hg <dig>
notes: for each tbp-odn-complex, ka is the association rate constant, kd is the dissociation rate constant, half-life  equals /kd, and k*d = kd/ka is the apparent dissociation constant.
tata-like subsequence: uppercase letters; Δg = -rtln is a change in gibbs free energy, where r =  <dig>  × 10- <dig> jk- <dig> is boltzmann's constant, t is temperature in degrees kelvin; the substitution corresponding to the minor allele of each snp is shown in boldface.
first, the above-mentioned results show that the variant -38a in the lep gene promoter increases the rate of formation of the tbp-dna complexes  by 50%, the variant -30t fivefold, and variant -35g by 24-fold.
the rate of decay  for -38a increased by 20%, and for -35g by 14-fold, whereas for -30t, it decreased  <dig> -fold.
affinity  of tbp for odn containing -38a increased by 50%, and for the -30t variant it increased by 12%, whereas for the -35g variant, the affinity diminished  <dig> -fold.
meanwhile, the minimal half-life of the complex  of  <dig> min was observed for the -35g variant; this situation was caused by the increase in the rate of day  14-fold relative to the norm and by very low affinity.
it should be noted that the variants -38a and -30t are located in the flanks of the tatabox , and only variant -35g affects the sequence of the tatabox itself, thereby causing a massive decrease in affinity to  <dig> nm.
as shown in table  <dig> the greatest increase in the rate of formation and decay of the tbp-tata complex was observed for variant -35g , which affects the sequence of the tata-box and replaces its most conserved base "a" with "g"  <cit> .
this change reduces affinity  to a virtually nonspecific level  and reduces half-life of the complexes more than sevenfold.
this alteration is associated with obesity  <cit> .
in contrast, variant -41g of the promoter of gcg lowers the rate of formation  of the complex tbp-odn  <dig> -fold, whereas the rate of dissociation  of the complex and its half-life decrease by 20%.
the calculated kd value, characterizing the change in tbp-odn affinity, decreased  <dig> -fold.
at the same time, their ratio, kd/ka, which determines the apparent dissociation constant k*d and affinity tbp-odn, decreased more than threefold in the case of -35g, which disrupts the tata-like subsequence  but increased  <dig> -fold for -38a flanking the normal a-38cta-35taa pentanucleotide.
we estimated the half-life of the tbp-odn complexes .
its values, 9- <dig> min, fit the time interval, 5- <dig> min , of nonequilibrium in vitro conditions for our measurement of the kinetics of tbp's binding to each odn corresponding to our three possible obesity-related snp markers.
the minimal half-life,  <dig> min, was observed for the allele -35g which corresponds to minimal binding affinity and to a maximal dissociation rate of the complex tbp-tata.
thus, judging by the effects of rs <dig>  rs <dig>  rs <dig>  and rs <dig>  the constants ka and kd describe qualitatively different and independent characteristics.
this observation is in agreement with the commonly accepted notion that k*d, ka, and kd independently characterize different features of the kinetics of intermolecular binding.
the different ranges of k0d and k*d reflect the differences in uncontrollable factors under equilibrium and nonequilibrium in vitro conditions, which do not influence the relative values of tbp affinity for different variants of the tbp-binding site .
because of this property of tbp-binding sites , which was proven empirically by many research groups  <cit> , leptin production in a patient with the minor variant -35g of rs <dig> may be proportionately lower  in comparison with a person carrying the variant wt.
consequently, the total plasma leptin level in this patient will be lowered: this is a clinically established risk factor of obesity  <cit> .
by the same token, the minor variants -38g of rs <dig> and -30t of rs <dig> may cause a proportional increase in leptin production  and a proportional increase in the overall plasma leptin level, which is a known risk factor of hypertension as a complication of obesity  <cit> .
this combined empirical and computer-based identification of rs <dig>  rs <dig>  rs <dig>  and rs <dig>  as obesity-related candidate snp markers is the main result of this work.
discussion
here, we focused only on the tbp-binding site because it is the best-studied site in human gene promoters  <cit> ; we detected known snp manifestations: "susceptibility/resistance" to diseases.
in comparison with our previous applications of the web service snp_tata_comparator  <cit>  to the research on monogenic diseases  <cit> , here we found a number of different associations, such as complications of obesity , a phenotypic trait , consequences of surgery , eating behavior , and the preventive effect of a lifestyle .
these findings can extend the practical utility of our web service because of the cross-validation of its output with a keyword search for available literature on an appropriate biochemical marker of the diseases under study.
in addition to the commonly used textual analysis of dna sequences, we take into account quantitative values of the minor-groove width of b-helical dna  <cit>  because tbp binds to dna via incorporation of side chains of two amino acid residues into dna's minor groove  <cit> .
this additional criterion increases the accuracy of our prediction , in agreement with independent findings of some other researchers  <cit> .
this result means that the computer-based snp analysis may be improved if various quantitative parameters of conformational and/or physicochemical features of b-helical dna  <cit>  are taken into account in addition to the textual data.
furthermore, population frequencies of the minor alleles of unannotated snp can serve as another information source that may improve the predictions of candidate snp markers of a disease.
accordingly, table s <dig>  characterizes all the snps under study in terms of the population frequencies taken from the " <dig> genome browser"  <cit> .
as one can see, these parameters vary from sample to sample in a wide range .
in addition, many biomedical snp markers still had no values of population frequencies within the framework of the " <dig> genome browser"  <cit>  .
moreover, many biomedical snp markers were not still documented by the database dbsnp  <cit>  , -51t→c , -35a→c ; see table s <dig>  additional file 1).
at present, the above-mentioned population frequency values correspond to ethnic groups in regional subpopulations  rather than cohorts of patients with certain risk factors of diseases--overweight, smoking, and alcohol intake--to prioritize the candidate snp markers according to the biomedical standards  <cit> .
this is why the type of biomedical standardization of whole-genome data available today may advance postgenomic predictive preventive personalized medicine  <cit> .
our in vitro assays  and in silico predictions  all indicate that the greatest changes in the leptin level may be expected for the minor allele -35g, which disrupts the tata-like tbp-binding site and decreases the tbp-dna affinity threefold.
in contrast, the minor alleles -30t and -38a flanking this site seem to enhance this affinity by 25% and 50%, respectively.
we have already observed this pattern empirically in a previous study, namely, three snp markers of β-thalassemia  within the well-known tata box decrease tbp's affinity for the human hbb gene promoter 10-fold, whereas another biomedical snp marker, -27a→t , yields only a 20% decrease  <cit> .
as for the smallest change in the binding affinity of tbp for the promoter variant -30t , one can see in table s <dig>  that the same or even smaller effects are observed for some biomedical snp markers, such as rs <dig> , the -35a→c substitution , rs <dig> , and the -35a→c substitution .
in addition, one can see in table  <dig> that the quantitative values of the gibbs free energy change  are an unreliable criterion for distinguishing between variants of odns binding to tbp.
in contrast, the kinetic constant ka of the association rate of the tbp-odn complex does it best .
this finding means that the binding of tbp to a promoter is under kinetic control rather than under thermostatic one.
therefore, additional empirical measurements can enrich the preliminary bioinformatics predictions on the candidate snp markers of diseases.
finally, our combined in silico and in vitro data may serve as a good rationale for clinical researchers who wish to validate promising candidates for snp markers.
the definitive proof of an snp as a clinical marker is demonstration of a significant difference in its frequency between patients and healthy people, with adjustments for various confounding factors such as the ethnic and gender composition of the regional subpopulation in question, lifestyle, living conditions, climate, environment, and expressivity and penetrance of the disorder under study  <cit> .
CONCLUSIONS
changes in the affinity of a transcription factor for a regulatory dna sequence are commonly an appreciable but not a crucial factor of a disease; for this reason, diverse clinical manifestations of the interactions between genetic and environmental factors are typical of polygenic diseases  <cit> .
in the case of metabolic syndrome  <cit> , obesity is a strong contributing factor  <cit>  in addition to genetic predisposition.
prevalence of obesity among overweight europeans exceeds 50% and results in elevated risk of respiratory failure, atherosclerosis, and heart failure  <cit> .
in this work, we identified  <dig> obesity-related candidate snp markers.
their validation in accordance with proper biomedical standards may help to solve the global problem of treatment of metabolic syndrome  <cit>  by means of the postgenomic predictive preventive personalized medicine  <cit> .

 metabolic syndrome, one of the main global challenges for modern health care, involves elevated risk of several interrelated disorders: obesity, ischemic heart disease, hypertension, insulin resistance, type ii diabetes mellitus, and dyslipidemia. it is commonly believed that obesity is among the key risk factors of metabolic syndrome. obesity affects quality of life and life expectancy and is associated with cardiovascular disorders, cancer, diabetes, and damage to kidneys and liver. it has also been reported that obesity correlates with reproductive disorders in women, prostate diseases in men, and elevated risk of congenital anomalies in children. the discovery that the leptin gene lep is "the obesity gene" shed light on the regulation of energy metabolism. leptin is a peptide hormone with molecular mass ~ kd. its secretion by adipocytes is directly proportional to adipose-tissue weight. recessive mutations in the leptin gene are strongly associated with obesity in mice and humans. leptin circulates with blood and regulates food consumption and energy demand of the brain. with a decrease in fat weight, the plasma leptin level decreases to increase appetite and suppress energy consumption until the recovery of fat weight. when fat weight increases, the level of circulating leptin increases and suppresses appetite until a fat weight reduction. initially, it was believed that leptin is produced mainly by adipocytes, but later it was found that leptin is also produced in such organs as the stomach, heart, and placenta. in addition to body weight control, leptin plays other physiological roles. it modulates vascular tone and blood pressure and enhances angiogenesis and calcification of vascular cells. numerous studies showed a direct correlation between blood leptin concentrations and blood pressure. elevated blood pressure contributes to atherosclerosis. recent studies on the development of obesity in male wistar rats uncovered an association of diet-induced fatty liver with insulin/leptin resistance. recent years have witnessed an increasing number of women with metabolic imbalance during pregnancy, with a variety of consequences; the mechanism of this metabolic imbalance is still poorly understood. the molecular pathways linking obesity to the above disorders are unclear mostly because of the large number of genes related to the nervous system, endocrine system, and metabolic system that regulate energy metabolism. the contribution of a single gene is estimated to be 1-6%, which is close to the statistical significance threshold with the current accuracy of these estimates. postgenomic predictive preventive medicine offers a way around this obstacle by taking into account numerous snp markers of various partially obesity-related complications in the above-mentioned or other disorders. analysis of snp markers, which show differences between an individual human genome and the reference human genome, as part of postgenomic preventive personalized medicine allows for effective treatment, improvement of treatment, and prevention of complications of treatment. genome-wide snp identification is the goal of the project genomes. the dbsnp database documents current results of this project; thus, the reference human genome, hg which is thought to contain the ancestral versions of all snps, is constantly refined. it is available in the ensembl database via the web service ucsc genome browser. ensembl contains data on gene knockouts in animals, and this information is helpful, for example, for reconstruction of perturbation networks of various disorders and for development of therapeutic strategies. computer-based analysis of hundreds of millions of unannotated snps in genomes that are documented in the dbsnp database may accelerate the search for biomedical snp markers. for this purpose, all the identified snps were mapped onto whole-genome maps of genes and onto protein-binding sites in dna that were predicted in silico and detected in vivo using chromatin immunoprecipitation, interchromosomal contacts, nucleosomes, transcriptomes either in health, during infection, disease, or after treatment. on the basis of these data, many web services such as tfbs, activity, is-rsnp, regulomedb, rsnp-mapper, raven, selex_db, funseq, apeg, featurescan, snpchiptools, snp-med, snap, funcisnp, spot, rsnp_guide, and chromos facilitate the search for candidate snp markers in terms of ranking of unannotated snps by their similarity to biomedical snp markers in accordance with projections of these snps onto whole-genome maps. according to the central limit theorem, the accuracy of this similarity-based search for candidate snp markers should increase as the number, diversity, representativeness, and completeness of genome-wide maps increase. due to this mainstream approach, the most impressive progress has been achieved with snps located in protein-coding gene regions because of the invariant types of disruption in both structure and function of the altered proteins regardless of the cellular conditions. on the other hand, the effects of snps that are located in regulatory regions of genes are still difficult to predict in silico. most studies in this field deal with regulatory snps in binding sites of tata-binding protein in the region upstream of the transcription start of various mrnas encoded in the human genome. for this reason, model animals with a null-mutation or a knockdown of tbp are always inviable. previously, we developed a computer-assisted method for estimation of significance of the difference between ancestral and minor snp variants in terms of their effects on gene expression. later, we confirmed the predictions of this method by our independent experiments in vitro under both equilibrium and nonequilibrium conditions of the electrophoretic mobility shift assay. then, we verified these empirical findings using a wide range of real-time assays, such as surface plasmon resonance, stopped-flow, and fluorescence resonance energy transfer. in addition, we utilized independent results of over experiments by others. on the basis of this comprehensive validation of the method, we designed the web service snp_tata_comparator for the researchers who would like to analyze certain changes in core promoters of human genes. in the present work, we cross-validated the output of two computer-based methods: dna sequence analysis using web service snp_tata_comparator and keyword search for articles on biochemical markers of obesity. near the tbp-binding sites of human gene promoters, we found obesity-related candidate snp markers, including rs, rs and rs in the human gene of leptin and rs in the human gene of glucagon. we characterized them empirically and in terms of quantitative estimates of the equilibrium dissociation constant. we then validated these estimates in terms of the apparent dissociation constant, k*d ≡ kd/ka, the ratio of the association rate constant, and the dissociation rate constant of the tbp-dna complex by means of emsa under nonequilibrium conditions in vitro. we found a significant linear correlation between k0d values predicted in silico and k*d values determined by measurements in vitro for the obesity-related candidate snp markers rs rs rs and rs which may be useful for postgenomic predictive preventive personalized medicine.  the results of in silico analysis of the known and candidate snp markers in the tbp-binding sites of the human gene promoters we applied our web service snp_tata_comparator to biomedical and candidate snp markers in the tbp-binding sites of the human gene promoters taken from our recent review. table s shows the results. the human il1b gene promoter contains the biomedical snp marker rs this snp is associated with greater body fat in older men, graves' disease, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, gastric ulcer and chronic gastritis, and major recurrent depression. it is a widely studied regulatory snp marker in the tbp-binding sites of human gene promoters. previously, we also studied this snp in vitro by emsa under both equilibrium and nonequilibrium conditions as well as in silico. the human apoa gene has the -35a→c substitution relative to the start of transcript number of this gene. this snp is inside a proven tata box. it is associated with obesity, fatty liver, and hematuria. using this snp marker of obesity, we validated the suitability of our web service snp_tata_comparator for analysis of obesity-related polygenic human diseases. the human nos gene contains an snp marker of epilepsy and resistance to malaria. it is a -51t→c substitution relative to the start of transcript number that causes nos overexpression according to clinical research. we have previously studied this snp in depth using emsa and our computer-based method. in this work, an additional keyword search pointed to new data suggesting that nos overexpression may be a biochemical marker of obesity. on the basis of this empirical observation, we propose the -51t→c substitution in the nos gene promoter as a candidate snp marker of obesity. the human pgr gene contains the biomedical snp marker rs causing de novo appearance of a spurious tbp-biding site along with an additional pathogenic transcription start site at position + relative to the normal tss for transcript number of the same gene. this snp is associated with endometrial cancer in obese women. it was previously analyzed by our computer-based method. here, using a keyword search, we found new clinical data showing that pgr overexpression is a biochemical marker of male breast cancer in obesity. with this in mind, we propose rs as a candidate snp marker of breast cancer in obese men. the human esr gene contains the snp marker of a primary esr2-deficient pt tumor that can transform into breast cancer without preventive treatment. an additional keyword search pointed to new clinical data showing that an esr deficiency can reduce the risk of obesity after ovariectomy. according to these data, we propose the snp s as a candidate marker of the low risk of obesity after ovariectomy. the human hsd17b gene contains an snp marker of breast cancer. nevertheless, using a keyword search, we found a new promising study suggesting that weight loss in hsd17b1-deficient obese postmenopausal women by means of the appropriate diet and/or exercise reduces the risk of obesity-related cancers. thus, we propose rs as a possible predictive snp marker of effectiveness of preventive anticancer treatment among obese postmenopausal women. the human mbl gene contains the snp marker of variable immunodeficiency, preeclampsia, and stroke caused by deficient expression of this gene as we have also predicted in silico and confirmed under both equilibrium and nonequilibrium conditions in vitro. a keyword search for "mbl " "deficiency," and "obesity" pointed to clinical findings that an mbl deficiency can increase the risk of obesity. therefore, rs may be a candidate snp marker of obesity. near this biomedical snp marker, we found one more snp: rs, and then, predicted that it can impair the expression of mbl too. this is why we propose rs as a candidate snp marker of obesity. the human f gene: its promoter contains a biomedical snp, namely, the -35a→c substitution relative to the start of transcript number of this gene. this snp is a marker of moderate bleeding caused by an f deficiency. an additional keyword search pinpointed the clinical finding of a statistically significant correlation between the total f level in obese patients with type diabetes mellitus and the risk of cardiovascular complications. hence, we suggest the -35a→c substitution within the human f gene promoter as a candidate snp marker of the low risk of cardiovascular complications in obese patients with type diabetes mellitus. near this biomedical snp marker, we found two unannotated snps, rs and rs that can cause overexpression of the f gene. on the basis of the correlation, we propose the two snps rs and rs as candidate markers of the high risk of cardiovascular complications in obese patients with type diabetes mellitus. the human f gene contains a known snp marker of obesity, myocardial infarction, and thromboembolism caused by f overexpression. the human hbb and hbd genes contain the best-studied tbp-binding sites that are altered by a number of snp markers of resistance to malaria and thalassemia, namely: rs rs rs rs rs rs and rs previously, we have analyzed most of them in depth using our computer-based method as well as using emsa under both equilibrium and nonequilibrium conditions in vitro. using a keyword search, we found new clinical data showing that a hemoglobin deficiency can serve as a biochemical marker of chronic inflammation in comorbidities of obesity. this finding allows us to propose rs rs rs rs rs rs and rs as candidate snp markers of inflammatory complications in obesity. near these biomedical snp markers, we found three unannotated snps: rs rs and rs our analysis predicted that they can cause a hemoglobin deficiency. consequently, we propose them as candidate snp markers of inflammatory complications of obesity. after that, we analyzed all unannotated snps in the region in the only known promoter of the human lep gene, which we selected due to friedman's discovery that the lep gene is the "obesity gene": ob ≡ lep. table s shows three unannotated snps--rs rs and rs34104384--which can alter tbp's affinity for this promoter according to our prediction. the first one in the list can reduce the tbp-promoter affinity, whereas the two others can increase this affinity. using a keyword search, we found literature data showing that the leptin deficiency is a biochemical marker of obesity, whereas leptin overexpression can serve as a marker of obesity-caused hypertension. this is why we propose three more candidate snp markers of obesity-caused hypertension. finally, we selected one more unannotated snp within the tbp-binding site of the promoter of the human gcg gene of glucagon because this snp's association with obesity has not yet been examined. similarly, we predicted a gcg deficiency and found that it is a biochemical marker of the resistance to obesity during a high-fat diet. accordingly, we propose rs as a candidate snp marker of obesity resistance during a high-fat diet. we reviewed all of the above findings and selected four of our obesity-related candidate snp markers--rs rs rs and rs183433761--for empirical biochemical verification using emsa under nonequilibrium conditions in vitro because these snps have not yet been verified experimentally on their possible association with obesity. the results of in vitro analysis of the four selected candidate snp markers the primary experimental data from the in vitro analysis of the four selected candidate snp markers are depicted in figure table shows these results for rs rs rs and rs in terms of the primary experimental ka and kd values that characterize the kinetics of tbp's binding to the ancestral and minor variants of the tbp-binding site in the only promoter of the lep gene that is documented in the human reference genome hg notes: for each tbp-odn-complex, ka is the association rate constant, kd is the dissociation rate constant, half-life equals /kd, and k*d = kd/ka is the apparent dissociation constant. tata-like subsequence: uppercase letters; Δg = -rtln is a change in gibbs free energy, where r = × 10- jk- is boltzmann's constant, t is temperature in degrees kelvin; the substitution corresponding to the minor allele of each snp is shown in boldface. first, the above-mentioned results show that the variant -38a in the lep gene promoter increases the rate of formation of the tbp-dna complexes by 50%, the variant -30t fivefold, and variant -35g by 24-fold. the rate of decay for -38a increased by 20%, and for -35g by 14-fold, whereas for -30t, it decreased -fold. affinity of tbp for odn containing -38a increased by 50%, and for the -30t variant it increased by 12%, whereas for the -35g variant, the affinity diminished -fold. meanwhile, the minimal half-life of the complex of min was observed for the -35g variant; this situation was caused by the increase in the rate of day 14-fold relative to the norm and by very low affinity. it should be noted that the variants -38a and -30t are located in the flanks of the tatabox, and only variant -35g affects the sequence of the tatabox itself, thereby causing a massive decrease in affinity to nm. as shown in table the greatest increase in the rate of formation and decay of the tbp-tata complex was observed for variant -35g, which affects the sequence of the tata-box and replaces its most conserved base "a" with "g". this change reduces affinity to a virtually nonspecific level and reduces half-life of the complexes more than sevenfold. this alteration is associated with obesity. in contrast, variant -41g of the promoter of gcg lowers the rate of formation of the complex tbp-odn -fold, whereas the rate of dissociation of the complex and its half-life decrease by 20%. the calculated kd value, characterizing the change in tbp-odn affinity, decreased -fold. at the same time, their ratio, kd/ka, which determines the apparent dissociation constant k*d and affinity tbp-odn, decreased more than threefold in the case of -35g, which disrupts the tata-like subsequence but increased -fold for -38a flanking the normal a-38cta-35taa pentanucleotide. we estimated the half-life of the tbp-odn complexes. its values, 9- min, fit the time interval, 5- min, of nonequilibrium in vitro conditions for our measurement of the kinetics of tbp's binding to each odn corresponding to our three possible obesity-related snp markers. the minimal half-life, min, was observed for the allele -35g which corresponds to minimal binding affinity and to a maximal dissociation rate of the complex tbp-tata. thus, judging by the effects of rs rs rs and rs the constants ka and kd describe qualitatively different and independent characteristics. this observation is in agreement with the commonly accepted notion that k*d, ka, and kd independently characterize different features of the kinetics of intermolecular binding. the different ranges of k0d and k*d reflect the differences in uncontrollable factors under equilibrium and nonequilibrium in vitro conditions, which do not influence the relative values of tbp affinity for different variants of the tbp-binding site. because of this property of tbp-binding sites, which was proven empirically by many research groups, leptin production in a patient with the minor variant -35g of rs may be proportionately lower in comparison with a person carrying the variant wt. consequently, the total plasma leptin level in this patient will be lowered: this is a clinically established risk factor of obesity. by the same token, the minor variants -38g of rs and -30t of rs may cause a proportional increase in leptin production and a proportional increase in the overall plasma leptin level, which is a known risk factor of hypertension as a complication of obesity. this combined empirical and computer-based identification of rs rs rs and rs as obesity-related candidate snp markers is the main result of this work. discussion here, we focused only on the tbp-binding site because it is the best-studied site in human gene promoters; we detected known snp manifestations: "susceptibility/resistance" to diseases. in comparison with our previous applications of the web service snp_tata_comparator to the research on monogenic diseases, here we found a number of different associations, such as complications of obesity, a phenotypic trait, consequences of surgery, eating behavior, and the preventive effect of a lifestyle. these findings can extend the practical utility of our web service because of the cross-validation of its output with a keyword search for available literature on an appropriate biochemical marker of the diseases under study. in addition to the commonly used textual analysis of dna sequences, we take into account quantitative values of the minor-groove width of b-helical dna because tbp binds to dna via incorporation of side chains of two amino acid residues into dna's minor groove. this additional criterion increases the accuracy of our prediction, in agreement with independent findings of some other researchers. this result means that the computer-based snp analysis may be improved if various quantitative parameters of conformational and/or physicochemical features of b-helical dna are taken into account in addition to the textual data. furthermore, population frequencies of the minor alleles of unannotated snp can serve as another information source that may improve the predictions of candidate snp markers of a disease. accordingly, table s characterizes all the snps under study in terms of the population frequencies taken from the " genome browser". as one can see, these parameters vary from sample to sample in a wide range. in addition, many biomedical snp markers still had no values of population frequencies within the framework of the " genome browser". moreover, many biomedical snp markers were not still documented by the database dbsnp, -51t→c, -35a→c; see table s additional file 1). at present, the above-mentioned population frequency values correspond to ethnic groups in regional subpopulations rather than cohorts of patients with certain risk factors of diseases--overweight, smoking, and alcohol intake--to prioritize the candidate snp markers according to the biomedical standards. this is why the type of biomedical standardization of whole-genome data available today may advance postgenomic predictive preventive personalized medicine. our in vitro assays and in silico predictions all indicate that the greatest changes in the leptin level may be expected for the minor allele -35g, which disrupts the tata-like tbp-binding site and decreases the tbp-dna affinity threefold. in contrast, the minor alleles -30t and -38a flanking this site seem to enhance this affinity by 25% and 50%, respectively. we have already observed this pattern empirically in a previous study, namely, three snp markers of β-thalassemia within the well-known tata box decrease tbp's affinity for the human hbb gene promoter 10-fold, whereas another biomedical snp marker, -27a→t, yields only a 20% decrease. as for the smallest change in the binding affinity of tbp for the promoter variant -30t, one can see in table s that the same or even smaller effects are observed for some biomedical snp markers, such as rs, the -35a→c substitution, rs, and the -35a→c substitution. in addition, one can see in table that the quantitative values of the gibbs free energy change are an unreliable criterion for distinguishing between variants of odns binding to tbp. in contrast, the kinetic constant ka of the association rate of the tbp-odn complex does it best. this finding means that the binding of tbp to a promoter is under kinetic control rather than under thermostatic one. therefore, additional empirical measurements can enrich the preliminary bioinformatics predictions on the candidate snp markers of diseases. finally, our combined in silico and in vitro data may serve as a good rationale for clinical researchers who wish to validate promising candidates for snp markers. the definitive proof of an snp as a clinical marker is demonstration of a significant difference in its frequency between patients and healthy people, with adjustments for various confounding factors such as the ethnic and gender composition of the regional subpopulation in question, lifestyle, living conditions, climate, environment, and expressivity and penetrance of the disorder under study.  changes in the affinity of a transcription factor for a regulatory dna sequence are commonly an appreciable but not a crucial factor of a disease; for this reason, diverse clinical manifestations of the interactions between genetic and environmental factors are typical of polygenic diseases. in the case of metabolic syndrome, obesity is a strong contributing factor in addition to genetic predisposition. prevalence of obesity among overweight europeans exceeds 50% and results in elevated risk of respiratory failure, atherosclerosis, and heart failure. in this work, we identified obesity-related candidate snp markers. their validation in accordance with proper biomedical standards may help to solve the global problem of treatment of metabolic syndrome by means of the postgenomic predictive preventive personalized medicine. 
obesity affects quality of life and life expectancy and is associated with cardiovascular disorders, cancer, diabetes, reproductive disorders in women, prostate diseases in men, and congenital anomalies in children. near the sites binding to tata-binding protein in human gene promoters, we found obesity-related candidate snp markers, including rs; rs; rs; rs; rs and rs; rs and rs; rs rs and rs; and rs rs rs rs rs rs rs rs rs and rs. validation of the candidate snp markers by proper clinical protocols appears to have a strong rationale and may advance postgenomic predictive preventive personalized medicine. we cross-validated the output of two computer-based methods: dna sequence analysis using web service snp_tata_comparator and keyword search for articles on comorbidities of obesity. clinical research can validate snp markers via genotyping of patients and demonstration that snp alleles are significantly more frequent in patients than in healthy people. the search for biomedical snp markers of interest can be accelerated by computer-based analysis of hundreds of millions of snps in the genomes project because of selection of the most meaningful candidate snp markers and elimination of neutral snps. using an electrophoretic mobility shift assay under nonequilibrium conditions, we empirically validated the statistical significance of the differences in tbp affinity values between the minor and ancestral alleles of out of the snps: rs rs rs and rs we also measured half-life, gibbs free energy change, and the association and dissociation rate constants, ka and kd, of the tbp-dna complex for these snps.
 obesity affects quality of life and life expectancy and is associated with cardiovascular disorders, cancer, diabetes, reproductive disorders in women, prostate diseases in men, and congenital anomalies in children. the use of single nucleotide polymorphism markers of diseases and drug responses can help physicians to improve treatment. clinical research can validate snp markers via genotyping of patients and demonstration that snp alleles are significantly more frequent in patients than in healthy people. the search for biomedical snp markers of interest can be accelerated by computer-based analysis of hundreds of millions of snps in the genomes project because of selection of the most meaningful candidate snp markers and elimination of neutral snps.  we cross-validated the output of two computer-based methods: dna sequence analysis using web service snp_tata_comparator and keyword search for articles on comorbidities of obesity. near the sites binding to tata-binding protein in human gene promoters, we found obesity-related candidate snp markers, including rs; rs; rs; rs; rs and rs; rs and rs; rs rs and rs; and rs rs rs rs rs rs rs rs rs and rs. using an electrophoretic mobility shift assay under nonequilibrium conditions, we empirically validated the statistical significance of the differences in tbp affinity values between the minor and ancestral alleles of out of the snps: rs rs rs and rs we also measured half-life, gibbs free energy change, and the association and dissociation rate constants, ka and kd, of the tbp-dna complex for these snps.  validation of the candidate snp markers by proper clinical protocols appears to have a strong rationale and may advance postgenomic predictive preventive personalized medicine. obesitygenepromotertata-binding proteintbpsingle nucleotide polymorphismsnptbp/promoter affinity changestatistical significancecandidate snp markerprediction in silicoverification in vitro22- june the 7th international young scientists school novosibirsk, russia 