hepatitis b virus ( hbv ) is a hepatotrophic virus that causes a major global health problem .
an estimated 2 billion individuals have been infected with hbv and approximately 350 million have the chronic disease .
although the mechanism of hbv pathogenesis remains elusive , host genetic factors are proposed to govern the pathology of disease progression or regression , along with viral and environmental factors .
nk cells are activated in the early response to infection , and there is substantial population variability in the rates of hbv infection .
killer cell immunoglobulin - like receptors ( kirs ) are members of the group of cell - surface molecules that activate or inhibit nk cell interaction with hla class i molecules on the surface of target cells .
although detailed genetic and functional analyses exploring kir influences on hbv in large cohorts are lacking , accumulating evidence supports that nk cell activation contributes to inflammation and liver injury during hbv infection both in hbv transgenic mice and in hbv infected patients [ 58 ] .
kirs are members of the immunoglobulin superfamily of receptors and are encoded on chromosome 19q13.4 .
the kir gene cluster comprises up to 16 highly homologous and closely linked genes and pseudogenes .
fourteen of them encode receptors triggering either inhibition ( 3dl13 , 2dl13 , 2dl5 ) or activation ( 3ds1 , 2ds15 ) and/or both ( 2dl4 ) , and 2 pseudogenes ( 2dp1 and 3dp1 ) do not encode cell - surface receptors .
encode for cell - surface receptors , while the remaining alleles , 2ds4 * 003/4/6/7/8/9 , carry a 22-base pair deletion in exon 5 , which causes a frame shift , yielding a truncated kir2ds4 protein with loss of the transmembrane and cytoplasmic domains of the full - length kir2ds4 protein . additionally , different combinations of kir genes generate inherited haplotypes that can be divided into 2 basic groups  a and b  on the basis of their gene content .
polymorphic kirs , which interact with hla class 1 , are largely inhibitory and exhibit substantial genetic diversity .
the result is a significant variation of nk cell repertoire between individuals and also between populations . as each kir
- ligand interaction may have differential effects on nk cell activation and inhibition , this diversity has important potential influences on the host response to infections .
genetic studies have demonstrated associations between specific kir - ligand combinations and the pathogenesis and progression of diverse viral infectious diseases [ 1217 ] .
there is still limited data on the relationship of kir genes and chronic hbv infection in the literature .
additionally , to our knowledge , no analogous study has been performed in our population thus far .
the aim of this study was to analyze whether inhibiting or activatory kir genes and different kir genotypes have an association in the progression of hbv infection in a turkish population .
this may help to explore some of the possible immune genes that could be important in predisposition to chronic hbv infection .
the patient groups consisted of 37 patients ( 17 male , 20 female ) with chronic hbv infection and 36 patients ( 15 male , 21 female ) in spontaneous remission state of hbv infection diagnosed at the department of infectious diseases of the cukurova university , balcal hospital . the diagnostic criterion for chronic hbv adopted in this study
the control group consisted of 85 healthy subjects ( 38 males and 47 females ) .
the ages ranged from 18 to 70 years ( mean , 43 years ) for the patients with chronic hbv , from 19 to 78 years ( mean , 42.6 years ) for the patients with spontaneous remission , and from 19 to 74 years ( mean , 42.5 years ) for the control group .
all individuals included in this study were from the cukurova region of turkey and they were all matched for ethnicity .
all the enrolled subjects had no serological evidence of hepatitis c virus , hepatitis d virus , and hiv infections , and had no other diseases such as diabetes , malignant tumor , or autoimmune diseases .
samples were collected after informed written consent that was obtained from all participants , and the study was approved by the cukurova university ethics committee .
diagnosis of chronic hbv infection was based on seropositivity for anti - hbv antibody using electrochemiluminescence immunoassay ( roche diagnostics , gmbh , mannheim , germany ) and the confirmation of hbv dna using light cycler 2.0 real - time polymerase chain reaction ( roche diagnostics , gmbh , mannheim , germany ) .
dna from a venous blood sample of each subject was extracted by dna isolation kit ( qiaamp dna blood mini kit , cat no : 51104 , qiagen vertriebs gmbh , vienna , austria ) .
genotyping of kir genes was performed using the multiplex kir - sso typing kit from tepnel lifecodes corporation ( ref : 545110r , ct , usa ) .
this product consists of a mixture of locus - specific oligonucleotide probes coupled to color - coded microspheres ( luminex corp ) and 2 pcr reactions for the amplification of kir - exons 4 , 5 , 7 , 8 , and 9 . to type each sample ,
pcr was performed and the product was hybridized with the sso - probe mixture using the manufacturer s protocol .
frequencies of group - a and -b kir haplotypes were deduced from the genotype data . in individuals carrying only kir3dl3 , 2dl3 , 2dl1 , 2dp1 , 3dp1 , 2dl4 , 3dl1 , 2ds4 and 3dl2 , a fixed - gene content characteristic of group - a haplotypes was considered carrying 2 copies of group - a kir haplotypes ( aa genotypes ) . if any of genes kir2dl2 , 2dl5 ,
3ds1 , 2ds1 , 2ds2 , 2ds3 and/or 2ds5 were present , then the genotype was considered as having b haplotype ( bx ) .
the percentage of each kir gene in the patient and control groups was determined by direct counting ( individuals positive for the gene / individuals tested per population100 ) .
differences between 2 groups in the distribution of each kir gene were estimated by 2-tailed fisher s exact test and p<0.05 was considered to be statistically significant .
the patient groups consisted of 37 patients ( 17 male , 20 female ) with chronic hbv infection and 36 patients ( 15 male , 21 female ) in spontaneous remission state of hbv infection diagnosed at the department of infectious diseases of the cukurova university , balcal hospital . the diagnostic criterion for chronic hbv adopted in this study
the control group consisted of 85 healthy subjects ( 38 males and 47 females ) .
the ages ranged from 18 to 70 years ( mean , 43 years ) for the patients with chronic hbv , from 19 to 78 years ( mean , 42.6 years ) for the patients with spontaneous remission , and from 19 to 74 years ( mean , 42.5 years ) for the control group .
all individuals included in this study were from the cukurova region of turkey and they were all matched for ethnicity .
all the enrolled subjects had no serological evidence of hepatitis c virus , hepatitis d virus , and hiv infections , and had no other diseases such as diabetes , malignant tumor , or autoimmune diseases .
samples were collected after informed written consent that was obtained from all participants , and the study was approved by the cukurova university ethics committee .
diagnosis of chronic hbv infection was based on seropositivity for anti - hbv antibody using electrochemiluminescence immunoassay ( roche diagnostics , gmbh , mannheim , germany ) and the confirmation of hbv dna using light cycler 2.0 real - time polymerase chain reaction ( roche diagnostics , gmbh , mannheim , germany ) .
dna from a venous blood sample of each subject was extracted by dna isolation kit ( qiaamp dna blood mini kit , cat no : 51104 , qiagen vertriebs gmbh , vienna , austria ) .
genotyping of kir genes was performed using the multiplex kir - sso typing kit from tepnel lifecodes corporation ( ref : 545110r , ct , usa ) .
this product consists of a mixture of locus - specific oligonucleotide probes coupled to color - coded microspheres ( luminex corp ) and 2 pcr reactions for the amplification of kir - exons 4 , 5 , 7 , 8 , and 9 . to type each sample ,
pcr was performed and the product was hybridized with the sso - probe mixture using the manufacturer s protocol .
frequencies of group - a and -b kir haplotypes were deduced from the genotype data . in individuals carrying only kir3dl3 , 2dl3 , 2dl1 , 2dp1 , 3dp1 , 2dl4 , 3dl1 , 2ds4 and 3dl2 , a fixed - gene content characteristic of group - a haplotypes was considered carrying 2 copies of group - a kir haplotypes ( aa genotypes ) . if any of genes kir2dl2 , 2dl5 ,
3ds1 , 2ds1 , 2ds2 , 2ds3 and/or 2ds5 were present , then the genotype was considered as having b haplotype ( bx ) .
the percentage of each kir gene in the patient and control groups was determined by direct counting ( individuals positive for the gene / individuals tested per population100 ) .
differences between 2 groups in the distribution of each kir gene were estimated by 2-tailed fisher s exact test and p<0.05 was considered to be statistically significant .
framework genes kir2dl4 , 3dl2 , 3dl3 , and 3dp1 were present in all of the samples ( figure 1 ) . among inhibitory kir genes , 2dl1 and 3dl1 had higher frequencies in all samples of chronic hbv patients , which were more than 78.4% . with the exception of kir2ds4 ,
frequencies of the remaining activating genes were all lower than 54.1% ( table 1 ) .
the most frequent genotype , found in 13.5% of patients , was aa180 genotype , which consists of only 1 activating gene 
other patients demonstrated the presence of more than 1 activating gene and thus were considered as bx ( 67.6% ) .
two different aa genotypes  aa180 ( 13.9% ) and aa1 ( 8.3% )  were found in the spontaneous remission group , which consists of only one activating gene  kir2ds4 ( figure 2 ) .
other individuals in this group demonstrated the presence of more than 1 activating gene and thus were considered as bx ( 76.8% ) .
framework genes kir2dl4 , 3dl2 , 3dl3 , and 3dp1 were also present in all of the samples ( figure 2 ) . among inhibitory kir genes , 2dl1 and 3dl1 had higher frequencies in all samples of chronic hbv patients , which were more than 80.6% . with the exception of kir2ds4 ,
frequencies of the remaining activating genes were all lower than 63.9% ( table 1 ) .
in the first step , frequencies of individual kir genes were compared between chronic hbv patients and patients with spontaneous remission ( table 1 ) .
in the second step , frequencies of individual kir genes were compared between the control group and the group composed of chronic hbv patients and patients with spontaneous remission ( table 2 , figure 3 ) .
the rate of inhibitory kir2dl3 ( p=0.0 ) and 3ds1 ( p=0.0 ) were higher in the healthy group than the group composed of chronic hbv patients and patients with spontaneous remission .
since the methods used for kir genotyping permitted distinguishing groups of alleles of activating kir2ds4 , we found that there were no statistically significant differences for 2ds4 * 001 ( full - length exon 5 ) and 2ds4 * 003/4/6/7/8/9 ( deletion in exon 5 ) alleles between the 2 groups ( p>0.05 ) .
additionally , there were no statistically significant differences between the rate of aa and bx genotypes of chronic hbv patients and patients with spontaneous remission and the control group ( p>0.05 ) .
framework genes kir2dl4 , 3dl2 , 3dl3 , and 3dp1 were present in all of the samples ( figure 1 ) . among inhibitory kir genes , 2dl1 and 3dl1 had higher frequencies in all samples of chronic hbv patients , which were more than 78.4% . with the exception of kir2ds4 ,
frequencies of the remaining activating genes were all lower than 54.1% ( table 1 ) .
the most frequent genotype , found in 13.5% of patients , was aa180 genotype , which consists of only 1 activating gene 
other patients demonstrated the presence of more than 1 activating gene and thus were considered as bx ( 67.6% ) .
two different aa genotypes  aa180 ( 13.9% ) and aa1 ( 8.3% )  were found in the spontaneous remission group , which consists of only one activating gene  kir2ds4 ( figure 2 ) .
other individuals in this group demonstrated the presence of more than 1 activating gene and thus were considered as bx ( 76.8% ) .
framework genes kir2dl4 , 3dl2 , 3dl3 , and 3dp1 were also present in all of the samples ( figure 2 ) . among inhibitory kir genes , 2dl1 and 3dl1 had higher frequencies in all samples of chronic hbv patients , which were more than 80.6% . with the exception of kir2ds4 ,
frequencies of the remaining activating genes were all lower than 63.9% ( table 1 ) .
in the first step , frequencies of individual kir genes were compared between chronic hbv patients and patients with spontaneous remission ( table 1 ) .
there were no statistically significant differences between these 2 groups . in the second step ,
frequencies of individual kir genes were compared between the control group and the group composed of chronic hbv patients and patients with spontaneous remission ( table 2 , figure 3 ) .
the rate of inhibitory kir2dl3 ( p=0.0 ) and 3ds1 ( p=0.0 ) were higher in the healthy group than the group composed of chronic hbv patients and patients with spontaneous remission .
since the methods used for kir genotyping permitted distinguishing groups of alleles of activating kir2ds4 , we found that there were no statistically significant differences for 2ds4 * 001 ( full - length exon 5 ) and 2ds4 * 003/4/6/7/8/9 ( deletion in exon 5 ) alleles between the 2 groups ( p>0.05 ) .
additionally , there were no statistically significant differences between the rate of aa and bx genotypes of chronic hbv patients and patients with spontaneous remission and the control group ( p>0.05 ) .
nk cells express multiple cell surface receptors , and during different infections different receptors are likely to be important .
kirs have a variegated expression pattern , and their complex genetics indicate that they are involved in generating population diversity in antiviral immune response .
previous studies have demonstrated several associations between various kir genes and/or their hla class i ligands in clinical progression of hbv infection [ 2022 ] and in the progression of this infection to hepatocellular carcinoma .
these associations could be due to the capacity of interactions of hla class i molecules -especially hla - c and hla - b- with kirs and activation of nk cells . in the present study
, we examined the genes encoding kir receptors and kir genotypes in chronic hbv patients , patients with spontaneous remission , and healthy controls in a turkish cohort .
our results showed that the frequencies of both kir2dl3 and kir3ds1 genes were much higher in healthy controls than in chronic hbv patients and patients with spontaneous remission .
this might indicate that the inhibitory kir2dl3 and activating kir3ds1 were possibly protector genes for hbv infection .
similarly , gao et al , in their study comparing 182 chronic hbv patients with 140 healthy controls , reported that kir2dl3 : hla - c1 homozygosity was protective against hbv infection   and that kir2dl1 : hla - c2 was associated with susceptibility to hbv infection . in contrast , zhi - ming et al showed that kir2ds2 and kir2ds3 are hbv - susceptive genes , whereas kir2ds1 , kir3ds1 , and kir2dl5 may be protective genes that facilitate the clearance of hbv   in a han chinese population .
population genetic analyses have revealed that the frequency and distribution of kir genes and haplotypes vary with ethnicity .
although zhi - ming s study and ours were done in different populations , the kir3ds1 gene was found as a protector gene in both studies . in a wider study , lu
et al analyzed kir genes in 150 patients with chronic hbv infection , 251 spontaneous resolvers , and 451 healthy controls .
they found a lower frequency of the a haplotype and higher frequency of the b haplotype in patients exposed to hbv infection compared with healthy controls , implying a susceptibility effect of the b haplotype . in our study , there were no statistically significant differences between the rate of aa and bx genotypes of chronic hbv patients and patients with spontaneous remission and the control group .
additionally , in a recent study , pan et al suggested the association of a combination of full - length form and 22 bp - deleted form of kir2ds4 ( kir2ds4/1d ) with hepatocellular carcinoma incidence in patients with chronic hbv .
previous studies showed that there are important similarities between inhibitory and activating kir frequencies of patients with hepatitis c and hbv infections , despite these viruses being phylogenetically unrelated .
khakoo et al reported that genes encoding the inhibitory nk cell receptor kir2dl3 and its ligand , hla - c1 , influence resolution of hepatitis c virus infection in caucasians and african americans with expected low infectious doses of hcv but not in those with high - dose exposure .
chronic hbv patients , patients with spontaneous remission of hbv infection , and healthy controls in our population demonstrate that kir2dl3 and kir3ds1 are important immuno - genetic markers in determining antiviral immunity . to explore the role of kirs in hbv infections
, the interrelation between kir genes , genotypes , and chronic hbv infection should be investigated in many different populations .
models based on both activating and inhibitory receptors and their ligand interactions could be generated for detailed genetic studies in wider groups .
thus , in order to confirm the present general results obtained in this study , functional studies and investigations in different populations are still needed to better understand this mechanism .
 backgroundkiller cell immunoglobulin - like receptors ( kirs ) are a family of inhibitory and activating receptors expressed by natural killer ( nk ) cells and regulate nk cell activity in the innate response against viral infections . 
 the aim of this study was to determine the possibility of kir genes and genotypes as a candidate for susceptibility to or protection against chronic hepatitis b virus ( hbv ) infection or spontaneous remission of the infection in a turkish cohort.material/methodsthe present study was carried out on 37 patients with chronic hbv infection , 36 patients in spontaneous remission of hbv infection , and 85 healthy subjects . 
 sequence - specific oligonucleotide probes analysis was used to investigate 16 kir genes . 
 all data were statistically analyzed by the fisher exact test.resultsthe rate of inhibitory kir2dl3 ( p=0.0 ) and 3ds1 ( p=0.0 ) were higher in the healthy group than the group composed of chronic hbv patients and patients with spontaneous remission . 
 there were no statistically significant differences between the rate of aa and bx genotypes of chronic hbv patients and patients with spontaneous remission and the control group ( p>0.05).conclusionsour results suggest that kir2dl3 and kir3ds1 genes could be protector genes for hbv infection and they could be important immuno - genetic markers in determining antiviral immunity in the turkish population . 
Background
Material and Methods
Study subjects and samples
Laboratory diagnosis of patients with chronic HBV infection
KIR genotyping
Prediction of group-A/-B KIR haplotypes
Statistics
Results
Distribution of KIR genes and genotypes in chronic HBV patients
Distribution of KIR genes and genotypes in patients with spontaneous remission
Relationship of KIR genes and genotypes with HBV infection
Discussion
Conclusions