non - alcoholic fatty liver disease ( nafld ) , the hepatic manifestation of metabolic syndrome , includes a spectrum of diseases ranging from simple steatosis , through steatohepatitis ( nash ) , to fibrosis and ultimately cirrhosis ( 1 , 2 ) . the disease definition and modalities used for diagnosis and epidemiology studies are not standardized ( 3 ) . the prevalence of nafld increased rapidly and it affects about 20% to 30% of the population in western countries ( 4 ) and 15% in china ( 5 ) . as a lipid metabolism disorder , nafld has a strong genetic component . a recent gwas from the genetics of obesity - related liver disease consortium identified lyplal1 rs12137855 for nafld in 7177 adults of european ancestry ( 6 ) . lyplal1 encodes lysophospholipase - like protein 1 , a 26 kda cytosol protein , which belongs to a subclass of lysophospholipase family ( 7 ) . recently , several single nucleotide polymorphisms ( snps ) , near human lyplal1 gene were revealed to be significantly associated with fat distribution in a relatively sex - specific pattern ( 8) , such as 3 snps including rs4846567 ( 9 ) , rs2605100 ( 10 ) and rs2820443 ( 11 ) near lyplal1 gene , which are associated with increased waist - hip ratio ( whr ) adjusted for bmi only in women not men . snp rs11118316 at lyplal1 is associated with visceral adipose tissue / subcutaneous adipose tissue ratio in both men and women ( 12 ) , and snp at rs12137855 near lyplal1 gene is strongly associated with nafld ( 13 ) . subsequent studies showed that lyplal1 plays an important role in fat distribution and lipid metabolism . lyplal1 rs12137855 , as the susceptibility gene of nafld , was widely studied , but the results were inconsistent . no research has been performed on the association between polymorphism of lyplal1 and nafld in chinese han population . the aim of our study was to investigate the association between nafld and lyplal1 in chinese han population and assess the effect of this gene on serum lipid profiles . the study was performed in accordance with the principles of declaration of helsinki and its appendices ( 14 ) . this study was approved by the ethical committee of qingdao municipal hospital ( qingdao , china ) and a written informed consent form was obtained from all patients before participation in the study . from may 2010 to may 2014 , we selected a total of 298 unrelated adult subjects , including 184 unrelated chinese patients of both genders and different ages ( 85 males , 97 females , mean age 43.18  11.53 years ) diagnosed with nafld and 114 healthy controls matched for sex and age ( 57 males , 57 females , mean age 40.77  11.47 years ) by b - type ultrasonography ( 15 ) . the subjects were collected from the department of gastroenterology and the medical center of qingdao municipal hospital . the diagnosis of nafld was performed under standard clinical evaluation conditions according to the aasid criteria . other causes of liver disease were excluded , including increased alcohol intake ( > 210/140 g / wk for males / females ) , as confirmed by at least one family member or friend and carboxydesialylated transferrin determination , viral and autoimmune hepatitis , hereditary hemochromatosis , and alphal - antitrypsin deficiency ( 16 ) . we excluded other related disease , such as subjects with type 1 diabetes mellitus and coronary atherosclerotic disease ( cad ) . the controls were confirmed as healthy by medical history , general examinations and laboratory examinations at the same hospital . blood samples of each subject for biochemical analyses were collected into ethylene diamine tetraacetic acid - containing tubes after an 12-hour overnight fast and the following information for each subject was gathered ; height , body mass , waist , hip circumference , calculating body mass index ( bmi ) equals to mass ( kg)/height ( m ) . environmental factors , such as diet and physical activity , were not recorded in this study . the blood sample tested for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) using routine enzymatic methods . serum alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) and -glutamyltransferase ( ggt ) concentrations were measured as previously described ( 17 ) . the genomic dna purification kit ( bioteke , biotechnology , beijing , china ) was used for extracting dna from peripheral blood following the manufacturer 's instructions and stored at 20c until use . genotyping for lyplal1 ( rs12137855 ) was performed by polymerase chain reaction ( pcr ) analysis using the following primers for lyplal1 polymorphism : 5'-tcctaagttcctattgtcccttca-3 ' and 5'-tgctgtggggtgagtca-3 ' . pcr amplification ( labnet , united states ) was performed as follows ; initial step of 95c for 10 minutes , followed by 35 cycles ; denaturation at 94c for 1 minute , annealing at 60c for 1 minute and elongation at 70 c for 1 minute . all pcr products were resolved using 2% agarose gel electrophoresis at 110 v for 30 minutes with a 237-base pair product in size . the lyplal1 genotypes were detected by direct dna sequencing using the abi prism sequence detection system abi3730 ( foster city , ca , usa ) . the genotyping call rate was more than 95% and the completion rate was > 99% . statistical analyses were performed using spss statistical software , version 17.0 for window ( spss inc . hardy - weinberg equilibrium between expected and observed genotype distributions was assessed using the  test . genotype and alleles were estimated by chi - square test and dna distributions between nafld patients and controls were analyzed by pearson s  test or fisher s exact test where appropriate . differences in characteristics between different groups were examined using student s t test , paired samples t - test or  test . the strength of the association between the polymorphism and nafld was evaluated by logistic regression analysis adjusted for confounders ( age , sex , smoking and hypertension , which were considered as continuous variables ) . the study was performed in accordance with the principles of declaration of helsinki and its appendices ( 14 ) . this study was approved by the ethical committee of qingdao municipal hospital ( qingdao , china ) and a written informed consent form was obtained from all patients before participation in the study . from may 2010 to may 2014 , we selected a total of 298 unrelated adult subjects , including 184 unrelated chinese patients of both genders and different ages ( 85 males , 97 females , mean age 43.18  11.53 years ) diagnosed with nafld and 114 healthy controls matched for sex and age ( 57 males , 57 females , mean age 40.77  11.47 years ) by b - type ultrasonography ( 15 ) . the subjects were collected from the department of gastroenterology and the medical center of qingdao municipal hospital . the diagnosis of nafld was performed under standard clinical evaluation conditions according to the aasid criteria . other causes of liver disease were excluded , including increased alcohol intake ( > 210/140 g / wk for males / females ) , as confirmed by at least one family member or friend and carboxydesialylated transferrin determination , viral and autoimmune hepatitis , hereditary hemochromatosis , and alphal - antitrypsin deficiency ( 16 ) . we excluded other related disease , such as subjects with type 1 diabetes mellitus and coronary atherosclerotic disease ( cad ) . the controls were confirmed as healthy by medical history , general examinations and laboratory examinations at the same hospital . blood samples of each subject for biochemical analyses were collected into ethylene diamine tetraacetic acid - containing tubes after an 12-hour overnight fast and the following information for each subject was gathered ; height , body mass , waist , hip circumference , calculating body mass index ( bmi ) equals to mass ( kg)/height ( m ) . environmental factors , such as diet and physical activity , were not recorded in this study . the blood sample tested for total cholesterol ( tc ) , triglycerides ( tg ) , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) using routine enzymatic methods . serum alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) and -glutamyltransferase ( ggt ) concentrations were measured as previously described ( 17 ) . the genomic dna purification kit ( bioteke , biotechnology , beijing , china ) was used for extracting dna from peripheral blood following the manufacturer 's instructions and stored at 20c until use . genotyping for lyplal1 ( rs12137855 ) was performed by polymerase chain reaction ( pcr ) analysis using the following primers for lyplal1 polymorphism : 5'-tcctaagttcctattgtcccttca-3 ' and 5'-tgctgtggggtgagtca-3 ' . pcr amplification ( labnet , united states ) was performed as follows ; initial step of 95c for 10 minutes , followed by 35 cycles ; denaturation at 94c for 1 minute , annealing at 60c for 1 minute and elongation at 70 c for 1 minute . all pcr products were resolved using 2% agarose gel electrophoresis at 110 v for 30 minutes with a 237-base pair product in size . the lyplal1 genotypes were detected by direct dna sequencing using the abi prism sequence detection system abi3730 ( foster city , ca , usa ) . the genotyping call rate was more than 95% and the completion rate was > 99% . statistical analyses were performed using spss statistical software , version 17.0 for window ( spss inc . chicago , il , usa ) . hardy - weinberg equilibrium between expected and observed genotype distributions was assessed using the  test . genotype and alleles were estimated by chi - square test and dna distributions between nafld patients and controls were analyzed by pearson s  test or fisher s exact test where appropriate . differences in characteristics between different groups were examined using student s t test , paired samples t - test or  test . the strength of the association between the polymorphism and nafld was evaluated by logistic regression analysis adjusted for confounders ( age , sex , smoking and hypertension , which were considered as continuous variables ) . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; ggt , gamma - glutamyl transpeptidase ; glu , glucose ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; nafld , non - alcoholic fatty liver disease patients ; tc , total cholesterol ; tg , triglyceride . data are presented as mean  sd . the genotypes distribution of lyplal1 was in accordance with the hardy - weinberg equilibrium in nafld and control groups ( pnafld = 0.323 ; pcontrol = 0.230 , respectively ) . to ensure the accuracy of our genotyping , we randomly repeated dna sequencing in 100 subjects for reverse sequencing . the genotype and allele distribution are shown in table 2 , which indicates no significant difference between the two groups ( p > 0.05 ) . the gene lyplal1 did not increase the risk of developing nafld ( or = 0.622 , 95% ci : 0.334 - 1.159 ) . p : nafld patients vs. control . to explore whether gene polymorphism affect the laboratory parameters , we compared non - carriers and carriers of variant allele ( rs12137855 ) in all subjects , nafld patients and healthy controls , respectively ( table 3 ) ; the results showed that there was a significant difference in weight , bmi and ldl . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; ggt , gamma - glutamyl transpeptidase ; glu , glucose ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; nafld , non - alcoholic fatty liver disease patients ; tc , total cholesterol ; tg , triglyceride . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; ggt , gamma - glutamyl transpeptidase ; glu , glucose ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; nafld , non - alcoholic fatty liver disease patients ; tc , total cholesterol ; tg , triglyceride . the genotypes distribution of lyplal1 was in accordance with the hardy - weinberg equilibrium in nafld and control groups ( pnafld = 0.323 ; pcontrol = 0.230 , respectively ) . to ensure the accuracy of our genotyping , we randomly repeated dna sequencing in 100 subjects for reverse sequencing . the genotype and allele distribution are shown in table 2 , which indicates no significant difference between the two groups ( p > 0.05 ) . the gene lyplal1 did not increase the risk of developing nafld ( or = 0.622 , 95% ci : 0.334 - 1.159 ) . to explore whether gene polymorphism affect the laboratory parameters , we compared non - carriers and carriers of variant allele ( rs12137855 ) in all subjects , nafld patients and healthy controls , respectively ( table 3 ) ; the results showed that there was a significant difference in weight , bmi and ldl . abbreviations : alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bmi , body mass index ; ggt , gamma - glutamyl transpeptidase ; glu , glucose ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; nafld , non - alcoholic fatty liver disease patients ; tc , total cholesterol ; tg , triglyceride . in the recent years , this gene has been widely studied , but the results were inconsistent ( 6 , 18 ) . speliotes et al . observed significant associations with both histologic nafld and ct nafld at variants or near lyplal1 ( 6 ) . our study for the first time investigated the association between lyplal1 rs12137855 and nafld in chinese han population ; we selected 184 nafld patients and 114 controls to observe the association between lyplal1 rs12137855 and nafld ; however , we did not find significant association between gene and nafld , which is in accordance with some previous findings ( 18 - 23 ) . multiple factors are involved in development and progression of nafld such as insulin resistance , obesity and oxidative stress ( 24 ) . in our study , we diagnosed nafld using routine blood testing and liver ultrasonography . lack of direct measurement of hepatic fat content by gold standard liver biopsy reduced the accuracy of diagnosis , but we observed that metabolism indicators change obviously . alt and ast are used as markers of liver fat accumulation ( 25 - 27 ) and commonly used in clinical practice ( 28 ) . we can observe significant differences in plasma concentrations of these transaminases between nafld and healthy controls . on the contrary , independent of genetic variation in lyplal1 , these results were not in accordance with paola leon - mimila and speliotes study ; they confirmed that lyplal1 rs12137855 was associated with increased tg content ( 6 , 29 ) . interestingly lyplal1-related proteins have been predicted to play a role in consecutive steps in triglyceride breakdown ( 30 , 31 ) . pnpla3 has been confirmed to increase hepatic steatosis through preventing the breakdown of triglyceride ( 32 ) . whether lyplal1 has the same function and knowing the mechanism of triglyceride breakdown need more investigations . for chinese subjects , bmi of 28 kg / m or more is an index of obesity ( 16 ) . in this study , kg / m ) than controls ( 23.04  3.47kg / m ) ( p < 0.05 ) , also higher in the variant carrier ( 25.29  3.51 independent of this gene in nafld group and control group the difference did not reach statistical significant . our study suggested that lyplal1 can influence bmi , which reflects the association with obesity indirectly . our findings for the first time found a significant difference between variant carriers and non - carriers regarding ldl ; the mechanism is not clear and needs further research . as far the studies on asian population were negative ( 18 , 19 ) , and we have reasons to doubt the correlation of the gene with nafld in asian population . to obtain more precise results , larger studies on multiple ethnic groups , such as asian indian or korean should be performed . our results may also be due to small sample size , ethnic differences in linkage disequilibrium ( ld ) patterns , ethnic - specific association and gene / environment interactions . this study provided preliminary evidence that there is no association between lyplal1 rs12137855 polymorphism and development of nafld in chinese han origin for the first time . further studies with large study samples and different ethnicity are needed to investigate the influence of this gene on nafld .