sarcoidosis is a multisystem inflammatory disease of unknown etiology , pathologically characterized by non - caseating epithelioid granuloma ( 13 ) . a definitive diagnosis of sarcoidosis is still challenging and no gold criteria for diagnosis
a set of non - specific clinical features and laboratory findings including hypercalcemia , hypergammaglobulinemia and high levels of angiotensin - converting enzyme ( ace ) , along with pathologic findings such as non - caseating granuloma are all considered to exclude other granulomatous diseases ( 4 ) .
genetic predisposition and exposure to the environmental factors may cause the disease and begin granulomatous reactions ( 5 ) .
access study ( a case controlled ethological study in sarcoidosis ) has demonstrated five exposures to sarcoidosis include farming , jobs raising birds , moulds and woodworking ( 6 ) .
however , these risk factors can vary in different races and geographical regions due to the genetic predisposition and exposure factors such as m. tuberculosis and mould .
for instance , hla - dqb1 * 06 has been associated with radiographic progression in an african - american cohort with advanced pulmonary disease ( 9 ) and uveitis in dutch cohort ( 10 , 11 ) . however , hla - drb1 * 07 , * 14 and * 15 were closely related to progressive pulmonary disease in a scandinavian cohort ( 12 ) .
meta - analysis studies promote a better understanding of the disease activity and its predictive risk factors . the serum ace ( sace ) , known as a biochemical marker of sarcoidosis , as high level of sace appears to be associated with the active form of the disease ( 13 ) .
relationship between sace level and ace genotype indicates that d / d genotype is related to the highest level and i / i with the lowest level of sace ( 1418 ) . in african - american cohort study in 1998 , the dd genotype was associated with increased risk of sarcoidosis , but was not confirmed by the next following studies in the same population ( 19 ) .
subsequently , there was no link between ace gene polymorphism and risk of sarcoidosis in german , dutch , italian , british , finish and czech populations ( 15 , 18 , 20 , 21 ) .
the aim of this study was to determine the distribution of ace i / d genotype among iranian patients with sarcoidosis in comparison to healthy subjects and to investigate the relationship between serum ace levels and the ace i / d genotype in these patients .
this descriptive cross - sectional study included 102 patients with sarcoidosis , from an ongoing sarcoidosis patient registry , who registered or referred to masih daneshvari hospital , a third - level hospital in tehran , iran .
sarcoidosis was diagnosed based on clinical and radiological evidences , along with histological and serological findings , and roll out other granulomatous diseases such as tuberculosis .
a control group of 192 non - dependent , age and sex matched individuals was recruited . in sarcoidosis patients and controls , those who suffered from diseases that increase or decrease
the peripheral blood was collected from the rest of patients and control subjects . the serum ace level at the first visit ( before taking any medication ) was measured .
this study was approved by the bioethics committee protocol of masih daneshvari hospital , tehran , iran .
pcr was used to identify i and d alleles , a segment from the intron 16 on ace gene that the size fractionation was detected by electrophoresis .
genomic dna was extracted from peripheral blood mononuclear cells ( pbmcs ) using qiagen dna extraction kit ( the qiaamp dna blood mini kit , germany ) according to the manufacture structure . in each pcr experiment , micro - tubes
were contained of 25 l pcr reaction consisted of 20 pmol/l of each forward and reverse primer : f 5 ctggagaccactcccatcctttct 3 and r 5 gatgtggccatcac - attcg tcagat3 , 10 l of 2x master - mix red , 10 l of extracted dna , and 3 l distilled water .
pcr condition was as the follows : an initial denaturation step for 2 min at 94 c , followed by 35 cycles of denaturation ( 30 sec at 94 c ) , annealing ( 30 sec at 65 c ) , extension ( 30 sec at 72 c ) , and the final extension step ( 72 c for 5 min ) ( abi thermal - cycler 5020 ) ( 23 , 24 ) .
only 10 l of the amplified pcr products were loaded on a 2% agarose gel .
the dna bands corresponding to 190bp as deletion and 490 bp as insertion were visualized by a gel doc viber transilluminator ( fig .
determination of genotypes i d , ii , and dd in sarcoidosis patients on 2% agarose gel : from left to right , gels contain marker followed by seven patients with sarcoidosis . the single lower band in patients a , d , e , f , and g corresponding to 190 bp indicates the dd type .
the genotype ii in patient b is also found as a single upper band corresponding to 490 bp .
the i d type as a double band , 490 and 190 bps is determined in c. nc= negative control the genotypes ii and i d in control subjects on 2% agarose gel : marker in lane m. the remaining lanes occupied by seven control subjects .
the remaining lanes contain double bands as the i d type . nc= negative control characterization of the study population as released as meansd with 95% ci and absolute frequencies for categorical variables were reported .
, chicago , il , usa ) and anova test were conducted to compare the genotypes sace after normalization by homogeneity of variance test .
the differences between the mean sace levels in different genotypes based on the normal distribution were analyzed using student s t - test .
genotypic and allele distributions in different groups were compared using the chi - square test .
the wald test was used to determine statistical significance for each of those independent variables .
the hosmer - lemeshow test was used to determine the goodness of fit of the logistic regression model . in all cases , standard
this descriptive cross - sectional study included 102 patients with sarcoidosis , from an ongoing sarcoidosis patient registry , who registered or referred to masih daneshvari hospital , a third - level hospital in tehran , iran .
sarcoidosis was diagnosed based on clinical and radiological evidences , along with histological and serological findings , and roll out other granulomatous diseases such as tuberculosis .
a control group of 192 non - dependent , age and sex matched individuals was recruited . in sarcoidosis patients and controls , those who suffered from diseases that increase or decrease
the peripheral blood was collected from the rest of patients and control subjects . the serum ace level at the first visit ( before taking any medication ) was measured .
this study was approved by the bioethics committee protocol of masih daneshvari hospital , tehran , iran .
pcr was used to identify i and d alleles , a segment from the intron 16 on ace gene that the size fractionation was detected by electrophoresis .
genomic dna was extracted from peripheral blood mononuclear cells ( pbmcs ) using qiagen dna extraction kit ( the qiaamp dna blood mini kit , germany ) according to the manufacture structure . in each pcr experiment , micro - tubes
were contained of 25 l pcr reaction consisted of 20 pmol/l of each forward and reverse primer : f 5 ctggagaccactcccatcctttct 3 and r 5 gatgtggccatcac - attcg tcagat3 , 10 l of 2x master - mix red , 10 l of extracted dna , and 3 l distilled water .
pcr condition was as the follows : an initial denaturation step for 2 min at 94 c , followed by 35 cycles of denaturation ( 30 sec at 94 c ) , annealing ( 30 sec at 65 c ) , extension ( 30 sec at 72 c ) , and the final extension step ( 72 c for 5 min ) ( abi thermal - cycler 5020 ) ( 23 , 24 ) .
only 10 l of the amplified pcr products were loaded on a 2% agarose gel .
the dna bands corresponding to 190bp as deletion and 490 bp as insertion were visualized by a gel doc viber transilluminator ( fig .
determination of genotypes i d , ii , and dd in sarcoidosis patients on 2% agarose gel : from left to right , gels contain marker followed by seven patients with sarcoidosis . the single lower band in patients a , d , e , f , and g corresponding to 190 bp indicates the dd type .
the genotype ii in patient b is also found as a single upper band corresponding to 490 bp .
the i d type as a double band , 490 and 190 bps is determined in c. nc= negative control the genotypes ii and i d in control subjects on 2% agarose gel : marker in lane m. the remaining lanes occupied by seven control subjects .
characterization of the study population as released as meansd with 95% ci and absolute frequencies for categorical variables were reported . statistical analysis using spss software version 16.0 ( spss inc .
, chicago , il , usa ) and anova test were conducted to compare the genotypes sace after normalization by homogeneity of variance test .
the differences between the mean sace levels in different genotypes based on the normal distribution were analyzed using student s t - test .
genotypic and allele distributions in different groups were compared using the chi - square test .
the wald test was used to determine statistical significance for each of those independent variables .
the hosmer - lemeshow test was used to determine the goodness of fit of the logistic regression model . in all cases ,
a 190bp fragment with the deletion ( allele d ) and a 490bp fragment with the insertion ( allele i ) for genotypes ii , i d and dd were analyzed in 102 patients and 192 controls ( fig .
serum ace activity in patients and controls with genotypes separation is described in table 1 .
the association between sace activity in three geno - types of sarcoidosis patients and the control subjects was significant .
dd and i d genotypes in controls and the patients with a maximum of mean value were observed . the significant difference between the mean of sace activity in sarcoidosis patients versus control groups ( p<0.0001 ) , and i d genotype ( p=0.002 ) and dd genotype ( p<0.0001 ) was observed by t - test .
2 show the relationship between the ace and three genotypes in a clustered box plot diagram .
genotypes and allele frequencies in sarcoidosis and control are shown in table 2 . in total , the genotype frequencies of ii , i d and dd in the patients with sarcoidosis was 17% , 35.5% and 47.1% , respectively , and the frequency of d allele was 0.65 .
the frequency of both dd and i d genotypes in sarcoidosis and control among female and male generally showed no significant difference ( p=0.05 ) by chi - square test . clustered boxplot diagram of the relationship between the ace level and ii - id - dd genotypes .
outliner numbers 42 and 43 in sarcoidosis patients with ii genotype and 45 in dd genotype of the controls were seen association of the ace i / d polymorphism with sace level in patients and controls ace genotype and allele frequencies in sarcoidosis patients compared with healthy controls p value of genotype between case and control groups , p - value of genotype between ii and ( i d or dd ) genotypes logistic regression analysis , adjusting age and gender ( table 3 ) showed that the i d genotype to genotype ii ( or = 0.35 , 95% ci = 0.170.73 , p=0.005 ) and the dd genotype to genotype ii ( or = 2.11 , 95% ci = 0.984.54 , p<0.05 ) for sarcoidosis disease could be considering as a predictor factor .
male gender does not reach statistical significance , and for the female gender - genotype ii / i d has been associated with a reduced risk of disease .
several studies have analyzed the relationship between ace gene i / d polymorphism and sarcoidosis disease .
the various regional and ethical studies have shown a significant correlation between ace genotypes and serum ace level ( 15 , 17 , 25 ) .
the present study with the statistical analysis of the relationship between angiotensin - converting enzyme ( ace ) genotype and sace level shows a significant association between ace gene polymorphism and sace activity in both patients with sarcoidosis and control subjects . in a japanese study ,
sace in comparison to controls showed a significant difference ( dd = 29 , 14% , i d = 101 , 48.8% , ii = 77 , 37.2% ) ( 18 ) .
the maximum sace level was found in dd group among both sarcoidosis patients and controls .
genotype distribution was the same in both patients and controls group . in the american cohort ,
detroit michigan , the ace genotype frequency was according i d > dd > ii order in african - american population patients with sarcoidosis . in caucasian population , the i d genotype was most detected in the patients and controls ( 26 ) .
another study from the uk and czech population , the genotype frequencies of i d , dd and ii was 47% , 28% and 25% , respectively in uk population . in czech population , the genotype frequency order was id > ii > dd in the patients with sarcoidosis ( 20 ) . in turkey cohort , as the same as our study , the predominant genotype was dd ( 45.7% ) , i / d genotype ( 42.8% ) , and i / i genotype was only seen in 11.4% of the patients .
the frequency of d allele is 0.65 ( p - value=0.06 ) that is much more comparable with the present study .
thus , the results of current study are very close to turkey report due to genetic affinity in this region , but the genotype frequency varies because of different populations . on the other hand , in turkey study , the frequency of i / d genotype ( 53.6% ) is higher in control subjects ( 27 ) .
therefore , in other studies ( 16 ) genotype distribution in patients and controls did not differ , whereas this frequency varies in our study and other studies such as the american cohort in african - american population .
the over - representation of d / d genotype in sarcoidosis patients and their families can be seen in comparison to controls ( 28 ) . on the other hand ,
attempts to show polymorphism and severity of chronic disease have been done ( 20 ) .
meanwhile , due to the nature of this disease , racial and regional differences in the polymorphism frequency , an association between sace activity and sarcoidosis exists
. therefore , to better understanding of the disease activity , its diagnosis and treatment , studies with the appropriate statistical scale from each region should be separately performed . in the current study
, we tried to address ace gene polymorphism , serum ace levels and its relationship to sarcoidosis with appropriate sample size .
the difference in sace levels in patients with sarcoidosis and controls was significant that is in accordance with previous findings in this field .
this level also in dd and i d genotypes showed significant differences between patients and controls .
the level of sace was higher in the dd genotype of both patient and control groups ( table 1 ) . in a systematic review ( 29 ) that is accordance with the present findings , the frequency of i d and dd genotypes in all cases and separately in males and females shows a significant difference ( table 2 ) . in this study , weighted mean dd / ii ratio was 1.85 for all studies , 2.01 for caucasians and 1.64 for asians ( 29 ) .
besides , this general finding is theoretically justified . the variation in the results of photometric measurements
polymorphism analysis can be considered in cases that sace will be higher than 97.5 percentile for dd or ii genotypes ( 29 ) . moreover , in the present study , the cases with the high level for sace in ii genotype patients are seen ( table 1 )
. the results can be helpful to obtain the sensitivity , specificity and cut - off value for sace and this outcome should be assessed , according to the region and race . at the end , we used binomial logistic regression analysis to show the role of genotype in the development of sarcoidosis .
prevalence of dd to ii genotype is 2.1-fold ( p=0.05 ) and this is less than the amount of the i d geno - type to ii genotype ( or=0.358 ) .
however , in women with the i d genotype versus ii , the risk of disease has decreased 0.3-fold . however
, this study has some limitations such as no clinical relationship with the sace level and ace genotype exists .
this can be helpful in the interpretation of the relationship between the disease and the sace level , meaning that how disease severity in different genotypes distributed and this fact that either ace genotype only effects on study result or severity of disease is important too .
although results of insertion / deletion polymorphisms and sace levels in different geographical areas do not follow the same pattern , these studies improve our knowledge about the i / d polymorphism , s sace levels and sarcoidosis in the future research , we attempt to to determine a cut off value for sace level and sensitivity and specificity of sace level , according to i / d genotype .
finally , we will introduce better models for diagnosis and prognosis of sarcoidosis by examining other aspects of genetics and cytokines .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .
 background : sarcoidosis is a multisystem inflammatory disease of unknown origin with characterization of small granulomas . 
 angiotensin - converting enzyme ( ace ) is a pathophysiologic marker of sarcoidosis . 
 we present the ace insertion / deletion ( i / d ) polymorphism in correlation with serum ace level in iranian patients with sarcoidosis.methods:from jan 2014 to jan 2015 , 102 iranian patients who histopathologically diagnosed for sarcoidosis and 192 healthy age and sex - matched controls were recruited . 
 pcr was used for detection of i / d polymorphism in ace gene.results:frequency of ii / id / dd genotype in sarcoidosis disease was 17% , 35.5% , and 47.1% , respectively . 
 the frequency of d allele was 0.65 . 
 a significant association between i / d genotypes and mean of sace level was seen ( dd=85.222.9 , p<0.001 ) . 
 more frequent genotype in sarcoidosis patients was dd ( 47% ) , i d genotype ( 45.9% ) was found more in controls . 
 logistic regression analysis adjusting age and sex showed that i d to ii ( or=0.35 , 95%ci=0.170.73 , p=0.005 ) and dd to ii ( or=2.11 , 95%ci=0.984.54 , p=0.05 ) could be considered as a predictor factor for the disease activity . 
 no significant model for men in sarcoidosis group was seen , while women with ii / id were associated with a reduced risk for the disease.conclusion:although more regional studies with appropriate statistical scale must be done to provide a better diagnosis and prognostic tool for this disease , this study demonstrates that i d and dd genotype could be predictive factors for sarcoidosis . 
Introduction
Materials and Methods
Samples
DNA extraction and PCR amplification
Statistical analysis
Results
Discussion
Conclusion
Ethical considerations