An Analysis Of Killer Cell Immunoglobulin-like Receptor And Human Leukocyte Antigen Gene-pair Frequencies In Hashimoto’s Thyroiditis Patients

Backgound:Killer cell immunoglobulin-like receptor (KIR) molecules modulate cell function by recognizing their putative ligands—HLA class I (HLA-C). Based on the presence of asparagine or lysine at position80of HLA-C molecule, HLA-C can be divided into two categories:HLA-C1(Asn80) and HLA-C2(Lys80), recognized by KIR2DL2/L3/KIR2DS2and KIR2DL1/KIR2DS1, respectively. Owing to interaction with HLA-C, KIR/HLA-C genotypes contributed to resistance to pathogens and reproductive success. In addition, different KIR/HLA-C genotypes send inhibitory or activating signals to regulate the activation of NK cells and subsets of T cells, which plays an important role in the pathogenesis of diverse diseases. Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease and is characterized by lymphocytic infiltrates within the thyroid glands. Both natural killer (NK) cells and T lymphocytes might be involved in the pathogenesis of HT.Aim:The aim of this study was to determine whether certain KIR/HLA-C genotype combination played a role in the pathogenesis of HT.Methods:1. Study population: The case group contained88unrelated individuals with HT who were diagnosed by criterion adopted in this study has been previously described. They were healthy donors at Shandong Province Hospital affiliated to Shandong University between2012and2013. Control group consisted of86randomly selected healthy subjects that contained males and females. All subjects had no known chronic disease and presence of other autoimmune diseases. This study was approved by ethics committee of Shandong provincial hospital affiliated to Shandong University and each of them had signed an informed consent for participating in this study.2. Genotyping: DNA was extracted from peripheral blood with a standard salting-out procedure. The KIR genotyping was performed by means of polymerase chain reaction/sequence-specific primers (PCR/SSP) in all recruited individuals for the following KIR genes:2DL1,2DL2,2DL3,2DL4,3DL2,3DL3,2DS1,2DS2, pseudogenes KIR2DP1KIR2DL4, KIR3DL2, KIR2DP1and KIR3DL3were used as positive markers of PCR. The sequence-specific primers used for the detection of KIR loci were based on primer sites that have been previously described.We performed HLA-C genotyping on the DNA of all samples. Genomic DNA was amplified using sequence-specific primers that have been previously described.3. Data analysis and statistical methods: Statistical analysis was performed using SPSS18.0program. The phenotypic frequency (pf) of each KIR gene was calculated as the percentage of positive numbers among all specimens. The pf frequency of KIR genes between HT and controls were tested by X2text (chi-square text) or Fisher’s exact test. In addition, KIR/HLA-C genotype complex differences were tested by the same way between two groups. Statistical tests were considered significant whenever p-value was less than0.05.Results:1. KIR/HLA-C complex frequencies in HT patients and controlsA panel of KIR2D/HLA-C gene combinations were explored in HT patients and control subjects (Fig.1). In all combinations, the frequency of KIR2DS2/HLA-C1was significant increased in patients compared to controls (36.36%vs15.12%,p<0.001), while no significant differences were observed in other KIR/HLA-C gene combinations between the two groups.2. Different frequencies of HLA-C genesWe chose88patients and86healthy controls to compare the HLA-C ligands of KIRs. We studied HLA-Cw01-08alleles, which are specific for KIR locus (Table2). HLA-C1(Asn80) consisted HLA-Cw01,03,07,08. and HLA-C2(Lys80) consisted of HLA-Cw02,04,05,06, The result showed that in HT patients HLA-Cw03and HLA-Cw08were significantly decreased when comparing to controls (15.91%vs40.70%,26.14%vs47.67%, p<0.01).3. We observed that frequency of combination of KIR2DL2/3/HLA-C1complex in the absence of KIR2DS2was decreased in HT patients than its corresponding cohorts. With this result, we probably inferred that KIR2DS2(-)2DL2/L3(+)HLA-C1(+) be the susceptive genotype in HT patients and suggested a preponderant protective effect of KIR2DL2/L3over KIR2DS2.Conclusion and significance:In HT patients, the combination of KIR2DS2and HLA-C1might influence NK cell activity and associate with the susceptibility of HT. The inhibitory combination of KIR2DL2/L3and their HLA-C ligand in the absence of relative activating KIR2DS2could be the susceptive genotype in HT.