the human apolipoprotein a - i gene ( apoa1 ) belongs to the apoa1-ciii - aiv gene cluster , located in chromosome 11q23 .
this cluster consists of evolutionarily related genes that regulate serum lipid and lipoprotein levels ( karathanasis , 1985 , elshourbagy et al . , 1986 ) .
the nucleotide sequence of apoa1 is interspersed by three introns ( karathanasis et al . , 1983 ) .
apoa1 codes for apolipoprotein a - i ( apo a - i ) . since apo a
- i is the major protein component of high density lipoprotein ( hdl ) particles which offer protection against atherosclerosis , therefore , much of the scientific work on apo a - i has traditionally focussed on its atheroprotective role .
but , recent studies have implicated derangements in serum apo a - i concentration in some other pathological conditions that are not conspicuously related to atherosclerotic disorders .
thus , genetic variations of apoa1 that influence serum apo a - i levels are of considerable interest .
the g-75a and c+83 t single nucleotide polymorphisms ( snps ) are two common variations of the apoa1 gene .
the g-75a polymorphism , located in the promoter region 75 base pairs ( bp ) upstream from the transcription start site of apoa1 , is due to a guanine to adenine interchange ( pagani et al . ,
the position of this snp has been variously described as   75  bp ,   76  bp and   78  bp .
this difference in representation is due to three different transcription start sites being described for apoa1 by different studies ( higuchi et al . , 1988 , sastry et al . , 1988 ) .
it is caused by a cytosine / thymine substitution in the position 83  bp downstream from the transcription initiation site ( wang et al . , 1995 ) .
each of these snps alters the recognition site for the restriction endonuclease mspi , which facilitates their detection by a restriction fragment length polymorphism ( rflp ) based technique .
historically , the g-75a and the c+83 t polymorphisms have been investigated in relation to coronary artery disease ( cad ) and cardiovascular risk factors ( reguero et al . , 1998 , jeenah et al . , 1990 , sigurdsson et al . , 1992 , talmud et al . , 1994 , wang et al . , 1996 , bai et al . , 1996 , chhabra et al . , 2003 , shanker et al . , 2008 , dawar et al . , 2010 , miroshnikova et al . , 2011 ,
however , it is increasingly believed that these polymorphisms may have important roles to play in a host of other disorders such as alzheimer 's disease ( vollbach et al .
, 2005 , smach et al . , 2011 ) , multiple sclerosis ( koutsis et al . , 2009 ) ,
schizophrenia ( yang et al . , 2010 ) , breast cancer ( hamrita et al . , 2011 ) , parkinson 's disease ( swanson et al . , 2014 ) , cholelithiasis ( dixit et al . , 2007 ) , gout ( cardona et al . , 2005 ) , diabetes mellitus ( ma et al . , 2003 ) and acute lung injury following cardio - pulmonary bypass surgery ( tu et al . , 2013 )
the g-75a and c+83 t polymorphisms have been studied globally in different populations . however , no information is available on these polymorphisms from the north - eastern region of india .
the distribution of these polymorphisms varies with the ethnicity of the studied population and geography as previous studies have indicated .
geographically , the north - eastern region of india is a unique corridor that links the indian subcontinent to east asia and southeast asia .
assam is the most populous and second largest ( in terms of land area ) of the eight north - eastern states .
data about the distribution of a polymorphism in the healthy subjects are a prerequisite to investigating the associations of the polymorphism with the implicated disease . with a renewed interest in g-75a and c+83 t polymorphisms worldwide and a variety of diseases being described in relation to them
, we attempted to describe these polymorphisms in a sample population from assam , northeast india for the first time .
our aim was to study the distributions of g-75a and c+83 t polymorphisms of the apoa1 gene in a cross - section of healthy subjects from assam , and further compare their frequencies with those observed in other populations from india and elsewhere .
additionally , we assessed the influence of these polymorphisms on the phenotype by determining the variation in fasting serum apo a - i concentrations across the detected genotypes .
a total of 150 unrelated and apparently healthy individuals of either sex were recruited for the study .
all the individuals were inhabitants of assam and identified themselves as natives of the state , with no history of migration from elsewhere at least in the past four generations .
the study was approved by the institutional ethics committee of gauhati medical college and hospital , assam .
all the subjects voluntarily provided informed written consent to participate in the study prior to enrolment .
blood samples ( after at least 12  h of overnight fast ) were drawn from the median cubital vein in two sets from each subject under sterile conditions .
one set consisted of 3  ml of blood anticoagulated in edta vials for isolation of dna and genotyping .
the second set included 3  ml of blood collected in plain vials and the serum separated by centrifugation after clot formation for apo a - i estimation .
genomic dna was isolated from peripheral blood leucocytes ( obtained from edta admixed whole blood ) following a rapid dna isolation protocol ( sambrook and russell , 2001 ) .
the concentration and purity of the genomic dna was ascertained by measuring the optical density ( od ) values at 260  nm and 280  nm .
the samples had acceptable purity with the od ratio ( 260  nm/280  nm ) in the range of 1.7 to 1.9 .
a 435  bp fragment at the 5 end of apoa1 gene spanning the promoter region and the first intron was amplified by polymerase chain reaction ( pcr ) using the following primer pairs as described previously ( larson et al . , 2002 ) : forward : 5-agg gac aga gct gat cct tga act ctt aag-3 , reverse : 5-tta ggg gac acc tac ccg tca gga aga gca-3 ( metabion international ag , germany ) .
the pcr reaction was performed with a 25  l reaction mixture ( 1  l dna template , 0.5  l each of forward and reverse primers , 12.5  l dreamtaq pcr master mix from thermofisher scientific , 10.5  l nuclease - free water ) in a thermal cycler ( bio - rad model s1000 , usa ) using the following cycling conditions : initial denaturation at 95  c for 5  min , denaturation at 94  c for 30  s , annealing at 62  c for 1  min , extension at 72  c for 30  s and final extension at 72  c for 10  min .
the amplified products were run on 1% agarose gels along with 100  bp dna ladder ( invitrogen , usa ) , and subsequently photographed under uv light using gel doc  xr  + system ( bio - rad , usa ) ( fig .
the presence of g-75a and c+83 t polymorphisms was ascertained using an rflp method by digesting the pcr product with mspi restriction enzyme . for this , 10  l of the pcr product was digested overnight at 37  c with mspi under conditions specified by the supplier ( new england biolabs inc .
the digestion products were analysed by running them on 12% polyacrylamide gels simultaneously with 50  bp dna ladder ( thermofisher scientific , usa ) . as the 435  bp pcr product contains three mspi cutting sites at    75 ,  +  37 and 
+  83 loci , its complete digestion would produce 4 fragments of sizes 66  bp , 114  bp , 46  bp and 209  bp .
+  83 loci are polymorphic and coincide with the g-75a and c+83 t snps respectively .
molecular sizes of the restriction fragments according to the genotype are shown in table 1 .
concentration of apolipoprotein a - i ( apo a - i ) in serum was determined by immunoturbidometry in vitros 5600 integrated system autoanalyser using commercially available reagents and kits ( vitros chemistry products , ortho - clinical diagnostics inc .
quality control measures were undertaken using control material ( apoa1 performance verifier 1 ) to ensure precision and accuracy of the test results .
all statistical analyses were performed using spss version 11.5 ( spss , chicago , usa ) . for continuous variables ,
the results were reported as means with standard deviation ( sd ) . the kolmogorov 
smirnov test was performed to ensure normal distribution of the data . for the qualitative variables ,
the allelic and genotype frequencies of the two snps and the linkage disequilibrium between them were determined using popgene 2.0 software ( yeh and boyle , 1997 ) .
conformity to hardy weinberg equilibrium ( hwe ) was assessed by goodness of fit chi - square test (  ) and g - square test .
the gender - wise frequencies of the polymorphisms were also calculated . besides , diplotype frequency of the snps was ascertained .
allelic frequencies obtained in our population were compared to those reported by other studies using  test .
comparison of continuous variables across genotypes was carried out by unpaired t - test or one way analysis of variance ( anova ) , whereas , categorical data were compared by  test ( with yates correction wherever required ) .
a p value of less than 0.05 was considered statistically significant and that less than 0.01 was considered highly significant .
a total of 150 unrelated and apparently healthy individuals of either sex were recruited for the study .
all the individuals were inhabitants of assam and identified themselves as natives of the state , with no history of migration from elsewhere at least in the past four generations .
the study was approved by the institutional ethics committee of gauhati medical college and hospital , assam .
all the subjects voluntarily provided informed written consent to participate in the study prior to enrolment .
blood samples ( after at least 12  h of overnight fast ) were drawn from the median cubital vein in two sets from each subject under sterile conditions .
one set consisted of 3  ml of blood anticoagulated in edta vials for isolation of dna and genotyping .
the second set included 3  ml of blood collected in plain vials and the serum separated by centrifugation after clot formation for apo a - i estimation .
genomic dna was isolated from peripheral blood leucocytes ( obtained from edta admixed whole blood ) following a rapid dna isolation protocol ( sambrook and russell , 2001 ) .
the concentration and purity of the genomic dna was ascertained by measuring the optical density ( od ) values at 260  nm and 280  nm .
the samples had acceptable purity with the od ratio ( 260  nm/280  nm ) in the range of 1.7 to 1.9 .
a 435  bp fragment at the 5 end of apoa1 gene spanning the promoter region and the first intron was amplified by polymerase chain reaction ( pcr ) using the following primer pairs as described previously ( larson et al . , 2002 ) : forward : 5-agg gac aga gct gat cct tga act ctt aag-3 , reverse : 5-tta ggg gac acc tac ccg tca gga aga gca-3 ( metabion international ag , germany ) .
the pcr reaction was performed with a 25  l reaction mixture ( 1  l dna template , 0.5  l each of forward and reverse primers , 12.5  l dreamtaq pcr master mix from thermofisher scientific , 10.5  l nuclease - free water ) in a thermal cycler ( bio - rad model s1000 , usa ) using the following cycling conditions : initial denaturation at 95  c for 5  min , denaturation at 94  c for 30  s , annealing at 62  c for 1  min , extension at 72  c for 30  s and final extension at 72  c for 10  min .
the amplified products were run on 1% agarose gels along with 100  bp dna ladder ( invitrogen , usa ) , and subsequently photographed under uv light using gel doc  xr  + system ( bio - rad , usa ) ( fig .
the presence of g-75a and c+83 t polymorphisms was ascertained using an rflp method by digesting the pcr product with mspi restriction enzyme . for this , 10  l of the pcr product was digested overnight at 37  c with mspi under conditions specified by the supplier ( new england biolabs inc .
the digestion products were analysed by running them on 12% polyacrylamide gels simultaneously with 50  bp dna ladder ( thermofisher scientific , usa ) . as the 435  bp pcr product contains three mspi cutting sites at    75 ,  +  37 and 
+  83 loci , its complete digestion would produce 4 fragments of sizes 66  bp , 114  bp , 46  bp and 209  bp .
the    75 and  +  83 loci are polymorphic and coincide with the g-75a and c+83 t snps respectively .
molecular sizes of the restriction fragments according to the genotype are shown in table 1 .
concentration of apolipoprotein a - i ( apo a - i ) in serum was determined by immunoturbidometry in vitros 5600 integrated system autoanalyser using commercially available reagents and kits ( vitros chemistry products , ortho - clinical diagnostics inc .
quality control measures were undertaken using control material ( apoa1 performance verifier 1 ) to ensure precision and accuracy of the test results .
all statistical analyses were performed using spss version 11.5 ( spss , chicago , usa ) . for continuous variables ,
the results were reported as means with standard deviation ( sd ) . the kolmogorov 
smirnov test was performed to ensure normal distribution of the data . for the qualitative variables ,
the allelic and genotype frequencies of the two snps and the linkage disequilibrium between them were determined using popgene 2.0 software ( yeh and boyle , 1997 ) .
conformity to hardy weinberg equilibrium ( hwe ) was assessed by goodness of fit chi - square test (  ) and g - square test .
the gender - wise frequencies of the polymorphisms were also calculated . besides , diplotype frequency of the snps was ascertained .
allelic frequencies obtained in our population were compared to those reported by other studies using  test .
comparison of continuous variables across genotypes was carried out by unpaired t - test or one way analysis of variance ( anova ) , whereas , categorical data were compared by  test ( with yates correction wherever required ) .
a p value of less than 0.05 was considered statistically significant and that less than 0.01 was considered highly significant .
we found both the g-75a and the c+83 t loci to be polymorphic in the current population .
the genotype and the allelic frequencies for the two polymorphisms are presented in table 3 .
all the three possible genotypes , namely , gg , ga and aa were detected .
the aa genotype was not found in females . on the other hand , for the c+83 t locus , the rare t allele frequency was 0.06 .
the distributions of g-75a and c+83 t polymorphisms were consistent with a population at hardy  weinberg equilibrium .
the genotype and allelic frequencies for the g-75a site with respect to the a allele was apparently lower in females than that in males , but , it was not statistically significant ( p  >  0.05 ) . however , for the c+83 t polymorphism , the frequencies were very similar in the two sexes .
we encountered 5 combinations of the two polymorphisms in the studied population : gg / cc , gg / ct , ga / cc , ga / ct and aa / cc ( fig .
overall , the gg / cc double homozygous variant was the most prevalent combination , while the double heterozygous ga / ct variant was the least common
table 5 compares the allelic frequencies of the two snps obtained in our study with those reported by other workers . to assess the effect of the two polymorphisms on apo a - i concentrations in serum ( table 6 )
this was done to take into account the gender - specific effects . for the g-75a snp ,
no significant differences ( p  >  0.05 ) in serum apo a - i values were noted across the gg , ga and aa genotypes .
likewise , serum apo a - i levels were comparable between the cc and the ct variants of the c+83 t locus too .
we further examined the combined influence of the two polymorphic sites by analysing the variation in serum apo a - i levels in a diplotype - specific manner .
however , the apo a - i values did not differ significantly across the 5 diplotypes ( p  >  0.05 ) .
the current study was undertaken to investigate the g-75a and c+83 t polymorphic sites of the apoa1 gene in a sample population from assam , northeast india from where data was hitherto not available .
both the polymorphisms were found to be in hardy  weinberg equilibrium , indicating that they are normally distributed in the study population . for the   75  bp site ,
the gg genotype was the most common in the study subjects , followed by ga and aa variants respectively .
however , it is unlikely that the aa variant is distributed in a sex - specific manner , and we attribute its absence in the female subjects of our sample to chance .
as all the aa homozygotes were men , the a allele frequency appeared to be higher in males ( 0.26 ) than in females ( 0.16 ) ; though statistically the difference was not significant .
for the +  83  bp locus , only the cc and the ct variants were detected .
the rarity of the tt genotype seems to be a universal phenomenon as it was not detected in a number of other populations too ( kamboh et al .
jia et al . , 2005 , dawar et al . , 2010 , biswas et al . , 2013 ,
when detected , the frequency of this variant was mostly found to be less than 1% ( sigurdsson et al . , 1992 ,
. , 2002 , miroshnikova et al . , 2011 , hamrita et al . , 2011 ) .
a review of existing literature reveals that only certain samples from northern india have exhibited a comparatively higher prevalence for this genotype ( dixit et al .
, 2007 , rai et al . , 2008 , khan et al . , 2012 ) .
the a allele frequency for the g-75a locus in our sample was found to occupy an intermediate position with respect to the other populations of the world .
nigerians had the lowest values ( ~  0.1 ) ( kamboh et al . , 1999 ) , while chinese had the highest values ( ~  0.3 ) ( heng et al . , 2001 , li et al . , 2008 ) in the spectrum .
our values were considerably higher compared to those of certain caucasian populations from europe ( jeenah et al .
, 1990 , sigurdsson et al . , 1992 , talmud et al . , 1994 ,
1996 ) , north america ( larson et al . , 2002 , ordovas et al . , 2002 ) and
brazil ( chen et al . , 2009 ) . when compared with populations from other asian countries in east asia and southeast asia regions , the malays ( heng et al . , 2001 ) and taiwanese ( chien et al . , 2008 ) were found to have a significantly higher frequency , whereas , the japanese ( bai et al . , 1996 ) had a significantly lower frequency of this allele .
koreans on the other hand had comparable values ( yang et al . , 2010 ) . amongst the indian studies ,
the a allele frequency was seen to be higher in north indians from new delhi ( dawar et al . , 2010 ) and kashmir ( khan et al . , 2012 ) as compared to our sample .
chandigarh ( rai et al . , 2008 ) were also found to have relatively higher values , though this was not statistically significant .
in contrast to the g-75a polymorphism , comparatively fewer reports are available on the c+83 t polymorphism .
the frequency of the rare t allele in our study was considerably lower than in nigerians ( kamboh et al . , 1999 ) and brazilians ( de franca et al .
, 2005 , chen et al . , 2009 ) , and higher than in non - hispanic whites ( kamboh et al . , 1996 ) .
it was also different from that reported in other indian populations from new delhi ( dawar et al , 2010 ) , kashmir ( khan et al . , 2012 ) , singapore ( heng et al . , 2001 ) , and mumbai and bangalore ( shanker et al . , 2008 ) .
the similarities in the allelic frequencies of the g-75a and the c+83 t polymorphisms seen amongst some populations may be due to ancestral homology .
likewise , the differences in the allelic frequencies observed across some other nationalities of the world are perhaps attributable to the diverse genetic background .
this holds true for the different values reported from various parts of india as well , since the indian population is heterogeneous .
the northeastern region of india is sandwiched between two major subcontinental regions  the indian subcontinent in the west and the east / southeast asia region in the east .
owing to the geographical proximity and strategic location , there have been multiple waves of migration to assam and the other northeastern states from the indian mainland and the neighbouring asian countries in the past .
the region has thus acted as a  melting pot for people of different stocks from the adjoining regions since the ancient times . by and large , the present day population of assam has an obvious affinity to the other indian populations , but with discernible mongoloid elements ( flatz et al .
the region represents a sort of ethnological transition zone between india and neighbouring china , myanmar and thailand ( ali and das , 2003 ) .
the rare allelic frequencies of the two apoa1 polymorphisms in the current population being recorded in the intermediate range between other indian ( chhabra et al . , 2003 , shanker et al . , 2008 , padmaja et al . , 2009 ) and southeast asian populations ( jia et al .
, 2005 , heng et al . , 2001 , li et al . , 2008 , chien et al . , 2008 ) are perhaps a reflection of the above fact .
indians in general have exhibited a lower frequency for the rare a allele as compared to their southeast asian counterparts .
for instance , a very high frequency of the a allele was observed in the control subjects by dawar et al .
but , this might not be truly representative because these control subjects were selected on the basis of absence of cardiovascular diseases , and hence may not necessarily represent the healthy population .
some studies have suggested that the g-75a polymorphism influences the apo a - i levels in serum .
the a allele has been found to be associated with significantly higher values of apo a - i in certain populations ( jeenah et al . , 1990 , talmud et al . , 1994 , sigurdsson et al .
, 1992 , rai et al . , 2008 , dawar et al . , 2010 , kamboh et al . , 1996 ) . however , in the current population , the serum apo a - i levels were comparable across the gg , ga and aa genotypes in males as well as in females .
our findings are in agreement with those observed in other populations where no effect of the a allele was noted on serum apo a - i levels ( dallinga - thie et al . , 1996 , heng et al . , 2001 , jia et al . , 2005 )
in contrast , in individuals belonging to the hei yi zhuang ethnic group in china , the gg homozygotes had higher serum apo a - i levels than the a allele carrying subjects ( li et al . , 2008 ) .
the c+83 t snp , which is situated in the first intron of apoa1 has been relatively less investigated as opposed to the g-75a snp .
although few population studies have shown this polymorphism to influence serum apo a - i concentrations ( kamboh et al .
, 1996 , dawar et al . , 2010 , larson et al . , 2002 ) , we observed no such effect .
the differences in serum apo a - i between the cc and ct genotypes were not significant in either sex of the current population .
, 2001 , de franca et al . , 2005 , jia et al . , 2005 , padmaja et al . , 2009 , chen et al . , 2009 ) . the proposed mechanism by which g-75a snp alters apo a - i expression is that the g to a substitution alters the transcription efficiency of the gene and the apo a - i production rate ( tuteja et al
this site is located in a gc rich sequence of the promoter which is subject to transcription regulation by binding of a 90  kda binding factor .
it is believed that the g to a interchange at the   75  bp site alters the binding affinity of this binding factor to the gc rich region and consequently influences the rate of transcription ( angotti et al . , 1994 ) . as for the c+83 t snp which is located in the first intron of apoa1 , it is a part of the cpg dinucleotide ( shemer et al . , 1990 ) .
the +  83  bp site and the 5-region of apoa1 are differentially expressed in tissues .
they are methylated in non - expressing tissues , but hypo - methylated in tissues that express the gene ( liver ) .
the cytosine to thymine transition at the +  83  bp site possibly results in further demethylation , and thus facilitates increased expression of apoa1 in the expressing tissues ( shemer et al . , 1990 , wang et al . , 1996 ) .
due to the contradictory findings reported by different studies , it is increasingly believed that the g-75a and the c+83 t polymorphic sites are probably in linkage disequilibrium with other regulatory elements that actually control the production of apo a - i .
for instance , strong linkage disequilibrium has been described between the g-75a polymorphism and the xmni polymorphism located in the 5-flanking region of the apoa1 gene ( paul - hayase et al . , 1992 ) .
the x2-allele of the xmni polymorphism was found at a higher frequency in hyperlipidemia than in normolipidemia ( kessling et al . , 1985 ) .
it may also be that the effects of these snps on the apo a - i values are seen only in certain ethnic groups . or the effects attributed to these snps in some of the earlier studies were actually due to gene 
another possibility is that they influence apo a - i production only in specific subgroups like females ( pagani et al . , 1990 , minnich et al . , 1995 ) .
considering the possibility that these two polymorphisms may influence the apo a - i concentrations in a synergistic manner , we further examined the variation in apo a - i values across the 5 diplotypes encountered in our study population .
still , none of the combinations seemed to be associated with higher apo a - i levels .
thus , irrespective of the gender and irrespective of the combined effect of the g-75a and the c+83 t snps , these two snps did not influence the apo a - i levels in the current population .
we described the distribution of these two polymorphisms in a healthy sample from assam , in northeast india for the first time .
we documented the differences and similarities in the allelic frequencies in the study population with respect to the populations in the adjoining regions and the other parts of the world .
we also observed that these polymorphisms do not directly affect the serum apo a - i levels in the studied population .
however , with the recent spurt in interest on the role of these two polymorphisms in a plethora of clinical conditions , it remains to be seen how these allelic variants influence various clinical outcomes in this population .
we hope our data will be useful for future studies investigating the role of these two polymorphisms in the implicated disorders in the northeast indian population .
the authors declare that they have no conflicts of interest , financial or otherwise associated with the manuscript .
 backgroundthere is a growing interest in the role of allelic variants of the apoa1 gene in relation to a number of disorders . 
 we described two common polymorphisms of the apoa1 gene , g-75a and c+83 t and investigated their potential influence on the serum apolipoprotein a - i ( apo a - i ) levels in the native population of assam  a region that is ethnically distinct and from where no information is hitherto available.methodsblood samples were collected from 150 healthy volunteers . 
 apo a - i levels were estimated by immunoturbidometry . 
 genotyping was done by a pcr - rflp method that involved dna extraction from whole blood , followed by polymerase chain reaction and digestion of the pcr product by mspi restriction enzyme , and analysis of fragment sizes in 12% polyacrylamide gel.resultsthe gg variant at g-75a locus and cc variant at c+83 t locus were the most prevalent . 
 gg / cc was the most common combination . 
 homozygous tt genotype was not detected in any of the subjects . 
 the rare allele frequencies for the g-75a and c+83 t sites were found to be 0.22 and 0.06 respectively , which significantly differed from those reported in some other populations in neighbouring regions . 
 serum apo a - i concentrations did not vary significantly across the detected genotypes . 
 these findings were consistent in both sexes.conclusionwe described the distribution of the g-75a and c+83 t polymorphisms of the apoa1 gene in the population of assam for the first time . 
 these polymorphisms were not found to directly influence apo a - i concentrations in this population either individually or synergistically . 
Introduction
Materials and methods
Study subjects
Blood sampling
DNA isolation and genotyping
Biochemical analysis
Statistical analysis
Results
Discussion
Conclusion
Conflict of interest