human abdominal aortic aneurysm ( aaa ) is the most common type of aneurysm in humans . it is characterized by abnormal dilation of the aorta and is associated with histopathological changes of the arterial wall . although the presence of atherosclerotic lesions is a common feature in aaa , the destruction of the arterial wall is a crucial point in the development of aneurysm . effective matrix macromolecules , such as elastin , collagen and glycosaminoglycans / proteoglycans , are responsible for the integrity of the arterial wall . hyaluronan ( ha ) is a large , nonsulfated glycosaminoglycan ( gag ) produced in the vasculature by smooth muscle cells and endothelial cells . it is synthesized at the inner face of the cell membrane by hyaluronan synthase ( has ) encoded by three genes to different chromosome localizations , followed by translocation to the outer surface and the intercellular space . recent in vitro investigations have shown that ha influences important cellular functions such as proliferation , migration and secretory capabilities . alterations in hyaluronan metabolism , distribution and function have been documented in many diseases e.g. arthritis , immune and inflammatory disorders , pulmonary and vascular diseases , as well as cancer . there seems to exist a delicately regulated balance between production and removal of ha that is central to its biological functions under normal conditions . we hypothesize that changes in ha content or aberrant variants of ha make the wall more susceptible to atherosclerotic stimuli , thus contributing to the aaa development . we therefore , carried out a case - control association study in order to verify the association between the 833a / g polymorphism , located on an exonic splicing enhancer motif of the has-1 gene , and risk of aaa . blood was collected from 146 aaa patients and 156 control subjects with a negative history of vascular diseases . controls were collected among volunteer healthy blood donors and among people hospitalized for traumatic accidents to avoid the use of hospitalized controls . all samples were collected in the cisanello hospital of pisa ( tuscany , italy ) . clinical diagnosis of aaa was confirmed by echographic and spiral computed tomographic ( ct ) scan evaluations . aaa was defined as a focal dilation of the abdominal aorta at least 50% larger than expected normal diameter ( antero - posterior and lateral aortic diameter values were increased to 50.7811.66 mm and 50.5413.35 mm , respectively ) . we recorded demographic and clinical data including age , gender , bmi ( body mass index ) , smoking habits ( s = smoker , ex = ex - smoker , ns = no smoker ; patients were considered ex - smokers if they had stopped smoking at least five years before inclusion in the study ) , the presence or absence of hypercholesterolemia ( defined as total plasma cholesterol level 220 mg / dl or cholesterol - lowering drug therapy ) , the presence or absence of hypertension ( defined as diastolic blood pressure > 95 mmhg and/or systolic blood pressure > 160 mmhg ) , the presence or absence of diabetes . genomic dna was isolated from edta blood samples by a modified salting - out procedure . allele - specific oligonucleotide pcr ( aso - pcr ) method was carried out for has-1 single nucleotide polymorphism ( snp ) 833 a / g genotyping in a 20 l reaction mixture and 1 u of taq polymerase ( eurobio - labtek , fr ) . primers of our own design were used : 5-gggttaaggatccgcacg-3 ( wild type allele ) , 5-gggttaaggatccgcaca-3 ( mutant allele ) and 5-ccttcctctcaagcatcagc-3 ( reverse ) for each reaction at 0.5 m . pcr was carried out for 40 cycles consisting of an initial denaturation step at 95c for 15 followed by 20 cycles at 95c for 1 , 68c for 30 ( 1c / cycle ) , 72c for 15 and to 20 cycles at 95c for 1 , 51c for 30 , 72c for 15 , with a final extension at 72c for 10. the 178 base - pair ( bp ) pcr product was visualized on a 2% agarose gel ( eurobio - labtek , fr ) stained with ethidium bromide . we tested the hardy - weinberg equilibrium of genotype distributions separately among case patients and control subjects by  test . we used unconditional multivariate logistic regression analysis to examine the association between the genetic polymorphism ( dominant model : heterozygotes+rare homozygotes vs. common homozygotes ) , and the risk of aaa . the calculation of the odds ratio and its 95% confidence interval was performed adjusting the analysis for the other covariates . a type - i error of 0.05 was used and all the tests were two - tailed . blood was collected from 146 aaa patients and 156 control subjects with a negative history of vascular diseases . controls were collected among volunteer healthy blood donors and among people hospitalized for traumatic accidents to avoid the use of hospitalized controls . all samples were collected in the cisanello hospital of pisa ( tuscany , italy ) . clinical diagnosis of aaa was confirmed by echographic and spiral computed tomographic ( ct ) scan evaluations . aaa was defined as a focal dilation of the abdominal aorta at least 50% larger than expected normal diameter ( antero - posterior and lateral aortic diameter values were increased to 50.7811.66 mm and 50.5413.35 mm , respectively ) . we recorded demographic and clinical data including age , gender , bmi ( body mass index ) , smoking habits ( s = smoker , ex = ex - smoker , ns = no smoker ; patients were considered ex - smokers if they had stopped smoking at least five years before inclusion in the study ) , the presence or absence of hypercholesterolemia ( defined as total plasma cholesterol level 220 mg / dl or cholesterol - lowering drug therapy ) , the presence or absence of hypertension ( defined as diastolic blood pressure > 95 mmhg and/or systolic blood pressure > 160 mmhg ) , the presence or absence of diabetes . genomic dna was isolated from edta blood samples by a modified salting - out procedure . allele - specific oligonucleotide pcr ( aso - pcr ) method was carried out for has-1 single nucleotide polymorphism ( snp ) 833 a / g genotyping in a 20 l reaction mixture and 1 u of taq polymerase ( eurobio - labtek , fr ) . primers of our own design were used : 5-gggttaaggatccgcacg-3 ( wild type allele ) , 5-gggttaaggatccgcaca-3 ( mutant allele ) and 5-ccttcctctcaagcatcagc-3 ( reverse ) for each reaction at 0.5 m . pcr was carried out for 40 cycles consisting of an initial denaturation step at 95c for 15 followed by 20 cycles at 95c for 1 , 68c for 30 ( 1c / cycle ) , 72c for 15 and to 20 cycles at 95c for 1 , 51c for 30 , 72c for 15 , with a final extension at 72c for 10. the 178 base - pair ( bp ) pcr product was visualized on a 2% agarose gel ( eurobio - labtek , fr ) stained with ethidium bromide . we tested the hardy - weinberg equilibrium of genotype distributions separately among case patients and control subjects by  test . we used unconditional multivariate logistic regression analysis to examine the association between the genetic polymorphism ( dominant model : heterozygotes+rare homozygotes vs. common homozygotes ) , and the risk of aaa . the calculation of the odds ratio and its 95% confidence interval was performed adjusting the analysis for the other covariates . a type - i error of 0.05 was used and all the tests were two - tailed . significant differences were observed in demographic factors between groups of aaa patients and control subjects , including age , gender , diabetes , hypertension , and smoking status ( table 1 ) . patients showed an elevated percentage of males , higher prevalence of diabetes and hypertension compared to controls . as expected , the group of aaa cases was older than controls . the genotype distributions for the has-1 polymorphism were consistent with the hardy - weinberg equilibrium ( p>0.05 ) . the simple analysis with a  test between the status ( case / control ) and has-1 genotypes revealed a trend in the co - dominant model ( three genotypes separated , p=0.077 ) and a statistically significant association in the dominant model ( p=0.042 , with yates ? however , the multivariate logistic analysis , after the adjustment for the above mentioned covariates , showed that the association between the risk of aaa and has-1 genotype was not statistically significant , as reported in table 2 . table 1demographic data.control ( n=156)aaa ( n=146)page ( years)55 ( 5069)74 ( 3094)<0.0001abmi ( kg / m)26.42.827.13.50.074male sex107139<0.0001hypertension1656<0.0001hypercholesterolemia22250.94diabetes2100.04s 18s 28smoking statusex 30ex 60<0.0001ns 49ns 25aaa : abdominal aortic aneurysm ; bmi , body mass index ; s : smokers ; ex : ex - smokers ; ns : no smokers . aaa : abdominal aortic aneurysm ; bmi , body mass index ; s : smokers ; ex : ex - smokers ; ns : no smokers . table 2adjusted odd ratios for the covariates reported in table 1.has1 genotypescasescontrolsodds ratio95% lower limit95% upper limitpcommon homozygotes1171071heterozygotes2141rare homozygotes300.650.133.150.5895% odds ratio ( or ) and 95% confidence intervals for the risk of aaa . or is adjusted for the covariates reported in table 1 . dominant model : the rare homozygotes are pooled with the heterozygotes . reference category : common homozygotes . 95% odds ratio ( or ) and 95% confidence intervals for the risk of aaa . or is adjusted for the covariates reported in table 1 . dominant model : the rare homozygotes are pooled with the heterozygotes . reference category : common homozygotes . its molecular weight is often very high mass , ranging from about 1010 da , depending upon the tissue , but it can also exist as smaller fragments and oligosaccharides under certain physiological or pathological conditions . originally thought to be an inert molecular  stuffing  in connective tissues , ha mediates , by specific receptors , many important functional activities e.g. cell motility , invasion and proliferation . indeed , several studies suggest that fragmentation of ha enhances its ability to activate cell - signaling pathways . ha is present in the extracellular matrix , on the cell surface and inside the cell . the notion that ha is likely to take part in the development of vascular pathologies has been promoted by a number of observations , which show marked changes in the content and distribution of ha in several arterial disease entities such as diabetic angiopathy , atherosclerosis and restenosis . early biochemical studies showed that ha concentration of human atherosclerotic plaques generally decreases with increasing severity of atherosclerosis . matrix molecules , such as elastin , collagen and glycosaminoglycans / proteoglycans ( gag/ pg ) , are all responsible for the integrity of the arterial wall . one of the most critical events implicated in the development of aaa , is the mechanical failure of collagen and elastin and although many investigations have focused on these components , our understanding of the metabolism of gag / pg in aaa is extremely limited . some studies on aaa report a decrease in the amount of ha that may be attributed either to ha - degrading enzymes released by inflammatory cells or to an altered synthesis of ha related to polymorphisms in the promoter gene . this latter hypothesis could be explained by the existence of splicing variants of has-1 that could activate specific inflammatory cell - signaling pathways and increase the protease production . in our study , a significant difference in the genotype distribution of has-1 polymorphism in aaa patients , compared to controls was not seen . we are aware that the present study is underpowered , however , our results show implicitly that the effect of the polymorphism is too weak to confer a decisive risk in the etiology of aaa . probably , other polymorphisms within has-1 could have a stronger impact on the risk of the disease .
the hyaluronan synthase 1 ( has-1 ) gene encodes a plasma membrane protein that synthesizes hyaluronan ( ha ) , an extracellular matrix molecule . 
 accumulating evidence emphasizes the relevance of ha metabolism in an increasing number of processes of clinical interest , including abdominal aortic aneurysm ( aaa ) . 
 the existence of aberrant splicing variants of the has-1 gene could partly explain the altered extracellular matrix architecture and influence various biological functions , resulting in progressive arterial wall failure in the development of aaa . 
 in the present study , we assessed the hypothesis that has-1 genetic 833a / g polymorphism could be associated with the risk of aaa by performing a case - control association study , involving aaa patients and healthy matched donors .