sarcopenia is defined according to the european consensus as a condition that involves a loss of muscle mass and declining strength and/or physical performance and has profound physiologic and clinical consequences , including but not limited to impaired protein turnover , mobility loss , osteoporosis , increased fracture risk , dyslipidemia , insulin resistance , overall frailty , and increased mortality . a necessary condition for a diagnosis of sarcopenia is therefore the extent of muscle mass loss that should be considered significant [ 1 , 2 ] . the combination of sarcopenia and obesity , defined as sarcopenic obesity , is an important public health that induces fragility in the elderly , and it is associated with functional limitations and increased mortality [ 4 , 5 ] .   in order to avoid the risk of sarcopenic obesity in the elderly and to prevent in the young population , the diagnosis of obesity requires the utilization of various methods , including body composition evaluation , metabolic , functional , and a genetic approach [ 69 ] .   a number of candidate genes for obesity   and osteoporosis have been identified , but the genetic basis of sarcopenia is still largely unknown . greater loss of muscle mass leading to sarcopenic obesity in women occurs increasingly with age . dxa - derived total body lean mass ( tblean ) measures can reflect both muscle mass and muscle strength , providing a reliable measure for assessment of sarcopenia and obesity [ 13 , 14 ] . de lorenzo et al . have identified the normal weight obese ( nwo ) syndrome , characterized by normal body weight and bmi , but high total body fat mass ( tbfat ) accumulation ( > 30% ) accompanied by tblean deficiency   and high level of atherogenic indices and oxidative stress [ 16 , 17 ] . the frequency of nwo syndrome in the worldwide population is near to 10% , and the prevalence is higher in women than in men [ 1821 ] . nwo had a higher prevalence of metabolic syndrome , dyslipidemia , hypertension ( men ) , cvd ( women ) , and a 2.2-fold increased risk of cvd mortality ( women ) compared with those with low tbfat . early inflammation and genetic predisposition characterizes the syndrome [ 7 , 2325 ] . moreover , a cross - talk between skeletal muscle and adipose tissue in nwo syndrome has been supposed and linked with the control of body weight , both fat stores and muscle mass . tumor necrosis factor alpha ( tnf- ) could play a decisive role in the proposed cross - talk between adipose tissue and skeletal muscle , with development of muscular abnormalities resulting in a loss of skeletal muscle mass and function . tnf- is a cytokine implicated in the metabolic disturbances of chronic inflammation with its biological actions including induction of insulin resistance , anorexia , and weight loss . it is overexpressed in adipose tissue , which is both a source of and a target for tnf- . the g / a 308 polymorphism in the tnf- promoter gene increases transcriptional activation of the tnf- gene . given the clinical significance of sarcopenic obesity , the purpose of the present study was to investigate the allelic frequency of the g / a 308 polymorphism in the tnf- promoter gene and to determine whether this polymorphism is associated with appendicular skeletal muscle mass index ( asmmi ) in nwo italian population . we comprehensively analyzed tbfat , tblean , anthropometric , and biochemical and cardiovascular risk factors parameters in tnf- gene association study in nwo subjects . the study was conducted on a sample of 120 italian caucasian women ( aged 20 to 60 ) , recruited from ongoing studies at the university of rome tor vergata ( italy ) , from june 2012 to january 2013 . exclusion criteria were exposure to hormonal treatment or development of secondary obesity due to endocrine diseases or serious intercurrent illness . subjects with a history or symptoms of coronary artery , cerebrovascular or peripheral artery disease , heart failure , renal or hepatic diseases , alcohol abuse , or smoking were excluded , as well as those with established type 2 diabetes . it was ascertained that none were taking lipid - lowering or antidiabetic medications , insulin , nitro derivatives , systemic corticosteroids , or any hormonal contraception . the subjects were categorized into 3 different groups according to bmi and the percentage of tbfat ( pbf ) : ( 1 ) normal weight ( nw ) women having a bmi < 25  kg / m , pbf < 30% ; ( 2 ) nwo women with bmi < 25  kg / m , pbf > 30% . nwo women had regular 28-day menstrual cycles ; ( 3 ) preobese - obese ( preob / ob ) women with a bmi > 25  kg / m , pbf > 30% . study design was clearly written in lay person language and provided to each study subject . a written informed consent was obtained from each patient and the study protocol conforms to the ethical guidelines of the declaration of helsinki . the collection of serum and dna as well as the experiments was approved , and the study was conducted according to the guidelines of the  tor vergata anthropometric parameters for all participants were performed by trained personnel ( body weight , height , hip , and waist circumferences ) , with participants wearing only light underwear and without shoes . waist and hip measures were taken using a flexible steel metric tape to the nearest 0.5  cm , with subjects standing with arms relaxed by their side and balanced on both feet . was measured to the nearest 0.1  kg , using a balance scale ( invernizzi , rome , italy ) . height ( m ) was measured using a stadiometry to the nearest 0.1  cm ( invernizzi , rome , italy ) . body mass index ( bmi ) was calculated as body weight ( in kilograms ) divided body height ( in meters ) squared . the total body composition was assessed by dxa ( idxa , g.e . medical systems , wi , usa ) , according to the previously described procedure . the cv value ( s.d./mean ) for repeated measurements is 1.02.2% in our laboratory depending on application . pbf was calculated as tbfat mass divided by total mass of tissues ( tbfat + tblean + bone mass )  100 . asmmi was calculated as follows = ( appendicular skeletal muscle mass / height ; women were defined sarcopenic with asmmi < 5.47  kg / m ) . all materials were immediately placed in ice and plasma was stored at 70c , for cytokine determinations . analyses were carried out according to the standard techniques by the accredited clinical chemical laboratories of the  tor vergata insulin resistance was determined by homeostasis model assessment - insulin resistance ( homa - ir ) = [ ( fasting glucose ( mmol / l )  fasting insulin ( iu / ml))/22.5 ] . atherogenic indices were calculated as follows : total cholesterol ( mg / dl)/hdl cholesterol ( mg / dl ) ( normal value < 5 for men and < 4.5 for women ) , ldl cholesterol ( mg / dl)/hdl cholesterol ( mg / dl ) ( normal value < 3.5 for men and <3 for women ) , and log ( triglycerides ( mg / dl)/hdl ( mg / dl ) ) ( normal value < 0.5 ) .   blood levels of tnf- cytokine were quantified with an elisa assay ( mabtech , italy ) according to the manufacturer 's instruction ( lower limit of tnf- detection was 1.6  pg / ml ) . sequencing of the 5-region ( 595 to + 390 ) of the human tnf- gene revealed a g to a a transition polymorphism at position 308 . digestion with ncoi enzyme , revealed the presence of two alleles : allele a1 gives two fragments of 87  bp and 20  bp , allele 2 a single 107  bp fragment . amplification of 100  ng of genomic dna was performed using initial denaturation at 94c for 4  min followed by 30 cycles of 95c for 1  min , 60c for 1  min , and 72c for 1  min with a final extension at 72c for 10  min . the buffer for pcr reaction contained 2.5  mm mgcl2 , 9.9  mm tris - hcl ( ph 8.8 ) , 50  mm kcl , 0.1% triton_x 100 , 0.200  m deoxyribonucleotide triphosphate ( dntps ) , 1  u of taq dna polymerase , and 0.2  m of each primer . the primer sequences are 5-aggcaataggttttgagggccat-3 and 5-tcctccctgctccgattccg-3. the amplified product was digested with ncoi and analysed on agarose gel and visualized under uv light after ethidium bromide staining . statistical analysis was performed using a computer software package ( spss for windows , version 13.0 ; spss , chicago , il ) . one hundred twenty caucasian italian women gave informed consent and were enrolled : 95 subjects were eligible for the study design according to inclusion criteria , with mean age of 33.61  12.01 years and mean bmi of 26.46  5.64 characterization of the nutritional status by bmi revealed that 3.16% ( n = 3 ) were underweight , 45.26% ( n = 43 ) were normal weight ( eutrophic ) , 26.32% ( n = 25 ) were overweight , and 25.26% ( n = 24 ) were obese . none of the subjects classified as obese by bmi were sarcopenic , according to asmmi . when all women were classified by dxa , the majority ( 82.10% , n = 78 ) were classified as obese , and 7.69% ( n = 6 ) of them presented sarcopenic obesity . significant difference in the diagnosis of obesity between bmi and pbf was observed with an error percentage of 30.39% ( p < 0.005 ) . characterization of sarcopenic obesity , determined by bmi or dxa , and the relative asmmi are presented in table 1 . there was no association with sarcopenic obesity independent of body composition determined by dxa . the baseline characteristics of subjects , classified according to their genotypes , were shown in table 2 . all subjects were successfully genotyped for the g / a 308 tnf- polymorphism , distinguishing in wild type homozygote ( g / g ) , heterozygosis ( g / a ) , and mutant homozygote ( a / a ) genotypes . a total of 70 subjects ( 73.68% ) defined as carrier (  ) group had the g / g 308 tnf- genotype with an average age of 33.64  12.05 years . 25 subjects ( 26.32% ) defined as carrier ( + ) group had the g / a 308 tnf- genotype with an average age of 33.24  12.29 years , without statistical differences in the mean age . there was a positive association between sarcopenia , sarcopenic obesity , and 308 tnf- genotype ; hence all the obese women with sarcopenia present sarcopenic obesity and were carrier (  ) ( data not show ) . the carrier ( + ) group did not have a worse metabolic profile than carrier (  ) group . only hs - crp ( g / dl ) values were significantly different between the two groups ( 0.73  1.78  g / dl versus 0.20  0.50  g / dl ; p  0.05 ) . the anthropometric , biochemical and body composition ( dxa ) data of nw , nwo , and preob / ob groups , in accordance with the genotype polymorphism at positions 308 of the tnf- gene promoter , were reported in table 4 . distinguishing population in 3 different groups we found n. 17 nw subjects ( 26.3  6.6 years , range 1939 ) ; n. 30 nwo subjects ( 29.7  9.4 years ; range 1759 ) ; n. 48 preob / ob ( 38.4  12.8 years , range 1759 ) .   significant differences between nw , nwo , and preob / ob subjects were observed . no significant differences in hc and w / h ratio , lean arms , lean legs , and lean trunk were observed between the nwo and nw women , whereas bmi , pbf , tbfat weight , and waist circumference were significantly different ( p < 0.001 ) . as expected , body weight , bmi , hc , wc , w / h ratio , pbf , tblean , and lean legs were significantly different between the preob / ob and nw groups . preob / ob subjects with respect to nwo showed higher weight , bmi , wc , hc , w / h ratio , pbf , and tblean ( p < 0.001 ) . respect to groups nw and nwo , preob / ob subjects showed higher amount of fat mass , evaluated as pbf ( p < 0.001 ) and tbfat ( p < 0.001 ) . moreover , preob / ob subjects showed higher sarcopenic index with respect to other groups ( p < 0.001 ) . in nw subjects no differences were observed between carrier (  ) and carrier ( + ) . however , significant inter and intragenotype differences between nw , nwo , and preob / ob in all evaluated parameters were remarked ( p < 0.001 ) . carrier ( + ) nwo subjects showed significantly higher hc with respect to carrier (  ) ( p < 0.001 ) . among overall carrier (  ) , nwo showed significant differences in lean arms , lean legs , lean trunk , and tblean with respect to nw and preob / ob ( p < 0.001 ) . however , these similar results have not been confirmed between carrier ( + ) ; in fact there are differences in terms of lean mass , but they are not significant . among overall carrier ( + ) , preob / ob showed higher weight , bmi , wc , hc , pbf , and tbfat with respect to nw and nwo ( p 0.05 ) and higher asmmi with respect to nw ( p 0.05 ) , whereas nwo showed higher bmi , pbf , and tbfat with respect to nw ( p 0.05 ) . regarding sarcopenic index values , among nw , 4.21% ( n = 4 ) of them were sarcopenic ( 50% were carrier (  ) and 50% carrier ( + ) ) ; the same percentage was observed in nwo subjects ( 100% carrier (  ) ) . moreover , in preob / ob , 2.10% of them were sarcopenic and all were carrier (  ) . no significant differences between nw and nwo were observed . with respect to nw and nwo , preob / ob subjects showed significantly higher levels of insulin ( p 0.01 ) , homa - ir ( p 0.01 ) , total cholesterol ( p 0.01 ) , ldl cholesterol ( p 0.01 ) , and triglycerides ( p 0.01 ) and lower levels of hdl cholesterol ( p 0.01 ) . regarding atherogenic indices , preob / ob showed higher values of tc / hdl ratio ( p 0.02 ) , ldl / hdl ratio ( p 0.02 ) , and log tg / hdl ratio ( p 0.03 ) and reduced values of tc / ldl ( p 0.01 ) than nw and nwo groups . preob / ob subjects showed higher levels of pcr than other groups ( p 0.01 ) . in preob / ob subjects we found slightly higher levels of tnf- than nw and nwo subjects ( p 0.01 ) . preob / ob carrier ( + ) genotype had significantly reduced levels of tnf- with respect to carrier (  ) ( p 0.03 ) . no statistical differences of tnf- circulating levels between nw and nwo were observed . obesity plays relevant pathophysiological role in the development of health problems , arising as result of complex interaction of genetic , nutritional , and metabolic factors . adipokines and myokines appear to be involved , respectively , in autocrine / paracrine interactions within adipose tissue and muscle , involved in the endocrine bidirectional cross - talk between these tissues . use of adiposity measures rather than bmi could be a better cardiovascular risk predictor and obesity - related diseases [ 33 , 34 ] . our results confirm these recent data , highlighting the problem of obesity misclassification by bmi , especially in women subjects . we confirmed that bmi significantly underestimates prevalence of obesity when compared to dxa direct measurement of percent body fat . the majority of women classified as normal by bmi were found to be obese by pbf . in our study , according to pbf classification , the overall percentage of obese women was 82.10% , with error percentage between the two classifications relevant ( 30.39% ) ( p < 0.005 ) . none of the subjects classified as obese by bmi were sarcopenic ; however when all women were classified by dxa , the 7.69% of them presented sarcopenic obesity . characterization of sarcopenic obesity and the asmmi revealed that 9.85% of the eutrophic subjects determined by bmi exhibited sarcopenic obesity , while none of the obese was sarcopenic . several genetic studies were performed in order to discover the genes involved in the development of obesity susceptibility , and more than 300 genes have been putatively identified . certain single nucleotide polymorhisms ( snps ) , occurring within the promoter region of a large number of genes , influence the transcriptional activity of genes , resulting in intraindividual differences in the amount of protein produced by the gene . tnf- has a direct paracrine function in adipose tissue , reducing fat mass through stimulation of lipolysis and reduction of lpl expression and activity .   in human skeletal muscle and adipose tissue , tnf- messenger rna levels were positively correlated with downregulation of the glut4 glucose transporter , body mass index ( bmi ) , fat mass , and serum levels of insulin and inversely correlated with lipoprotein lipase activity . the promoter region of the tnf- gene contains several single nucleotide polymorphisms ( snps ) , such as g / a 238 , g / a 308 , and c / a 863 , altering transcriptional activity of tnf- mrna . the functional role of the g / a 308 tnf- polymorphism is currently unclear .   in the present study , the genetic variation of the g / a 308 polymorphism in the tnf- promoter gene was firstly analyzed in nwo syndrome , comparing nw and preob / ob subjects . our data partially match the previously published data [ 16 , 2325 ] . in our subjects several studies showed that g / a polymorphism is not useful to promote obesity or metabolic abnormalities . in italian and danish population , g / a polymorphism is unlikely to play an important role in the development of obesity and its related metabolic abnormalities [ 39 , 40 ] . the allele a was associated with obesity , in individuals between 50 and 60 years . our result obtained in a young population confirmed the previous data [ 29 , 42 ] . in spanish obese subjects ( 70% females ) any differences of anthropometric , metabolic profile and adipokines levels in accordance with g the conflicting data can be due to differences in methodologies of study , suggesting that obesity could be related to this polymorphism after the fifth decade of life .   in our study , no differences in allele frequency of g / a 308 tnf- polymorphism were observed in nw , nwo , and preob / ob subjects , as well as no differences in serum levels of tnf- cytokine in the study groups with the exception of preob / ob carrier ( + ) . moreover , homa - ir values were significantly higher in all and preob / ob carrier (  ) subjects compared to nw and nwo women . however , no differences were observed between the homa - ir of nw and nwo women . one of the aftermath of insulin resistance is sarcopenia . in this study , we found significant associations with tblean and the tnf- gene polymorphism . to understand the role of the proinflammatory cytokines in skeletal muscle differentiation , which appear to be controversial [ 43 , 44 ] , it is important to clarify the several mechanisms of tnf-.   tnf- promotes the activation of transcription factors such as nuclear factor-b ( nf-b ) and ap-1 ( activator protein-1 ) . transforming growth factor--activated kinase-1 ( tak-1 ) is critical for tnf--induced activation of nf-b and mitogen - activated protein kinases ( mapk ) signaling pathways , especially p38 [ 45 , 46 ] . tak-1 connects tnf- to activin signaling , explaining how these cytokines can inhibit myogenesis , establishing the physiologic relevance of this pathway in the impaired regeneration observed in sarcopenia . the induction of activin a secretion downstream of cytokine pathway stimulation suggests a mechanism explaining how cytokines alter muscle differentiation , because it is well established that tgf- family members such as myostatin and activin a can block myoblast differentiation . moreover , tnf- drastically increases the production of matrix metalloproteinase ( mmp)-9 , leading to skeletal muscle tissue loss in various pathological conditions . this is the first study that shows the importance of the tnf- gene polymorphism to tblean variation in nwo syndrome , representing our pilot effort to further studies . finally , we mention some limitations of the study : ( 1 ) our study was performed only in adult female subjects , excluding elderlies ; ( 2 ) the number of included subjects was relatively low ; thus validation of the results in larger and independent cohorts is recommended to confirm our hypothesis of etiology of sarcopenic obesity susceptibility , depending on body composition , bmi , and tnf- gene polymorphism and to exclude the risk of false negative findings . these findings may be used to develop a prediction model for sarcopenia ; if confirmed in a larger population , it could represent a new management approach for the correct diagnosis and therapeutic choice in the prediction and prevention of sarcopenic obesity .
background and aim . 
 normal weight obese ( nwo ) syndrome is characterized by normal body mass index ( bmi ) , but high amount of fat mass and reduced lean mass . 
 we evaluated allelic frequency of the g / a 308 tnf- polymorphism and prevalence of sarcopenia in nwo . methods . 
 we enrolled 120 italian healthy women , distinguished into 3 groups : normal weight ( nw ) ; nwo , and preobese - obese ( preob / ob ) and evaluated anthropometric parameters , body composition by dual x - ray absorptiometry , blood tests , and genotyping of g / a 308 tnf- polymorphism . 
 results . 
 we found a positive association between sarcopenic obesity and 308 tnf- polymorphism . 
 all obese women were sarcopenic and were no carrier of mutation ( g / g ) . among all g 
 / g , nwo showed significant differences in lean mass and total body lean mass ( tblean ) with respect to nw and preob / ob ( p < 0.001 ) . regarding appendicular skeletal muscle mass index values , 
 4.21% of nw were sarcopenic ( 50% g / g and 50% g / a ) ; the same percentage was observed in nwo subjects ( 100% g / g ) . 
 moreover , 2.10% of preob / ob were sarcopenic and all were g / g . conclusion . 
 our study suggests that tnf- polymorphism contributes to sarcopenic obesity susceptibility , in association with body composition . 
 this is the first study that shows the importance of tnf- polymorphism to determine tblean variation in nwo syndrome .