Evaluation of sarcoidosis genetic risk based on 18 susceptibility markers in a West-Slavonic population: subanalysis in context of Lӧfgren syndrome
M. Petrek (Olomouc, CZ)
Sarcoidosis is a multi-systemic inflammatory disease, affecting mostly lungs. The etiology is unknown and its disease course is variable. The sarcoidosis sub-phenotype Lӧfgren syndrome (LS) is characteristic by acute onset and better prognosis. The disease is suggested to be influenced by environmental factors and genetic susceptibility. Several genes involved in immune responses have been reported, suggesting pulmonary sarcoidosis as a polygenic disorder. In this study, we analysed 18 SNPs among 564 sarcoidosis patients, its sub-phenotypes LS (n=94) and non-LS (n=414) in comparison to 301 healthy controls from West-Slavonic population (Czech Rep. and Poland). Genotyping was performed using MALDI-TOF mass spectrometry based MassARRAY iPLEX platform (Agena Bioscience, San Diego, CA). Association analysis using allelic model and odds ratios (OR) was estimated using Fischer’s exact test; all significance statements and p values are given after Bonferroni correction for multiple comparisons. Analysis of sarcoidosis cases revealed association of total 7 variants associated with sarcoidosis as a whole. Analysis for sub-phenotypes LS/non-LS showed four variants specifically associated with Lӧfgren syndrome: HLA-DQA rs2187668*T (OR = 3.14, p = 1.09x10-6); HLA-DRA rs3135394*G (5.23, 8.25 x10-13); TNFalpha rs1800629*A (2.66, 5.94x10-7); LRRC16A rs9295661*C (2.97, 4.29x10-4). One variant was specifically associated with non-LS: ANXA11 rs1049550*A (0.66, 2.71x10-4). Our results replicate the findings from large cohort studies performed mostly in western Europeans and extend them to the Polish population, which has not been investigated in this context. Characterization of possible predisposing gene variants for sarcoidosis, and its sub-phenotypes such as Lӧfgren syndrome could be helpful for diagnosis of acute onset of the disease.
The influence of HLA-Shared Epitope (SE) and smoking in CCP autoimmunity in Greek patients with Rheumatoid Arthritis
K. Tarassi (Athens, GR)
Rheumatoid Arthritis (RA) is a complex, multifactorial autoimmune disease, whose etiopathogenesis involves genetic and environmental factors. The aim of the study was the assessment of association of HLA-DRB1*-SE in the presence/absence of CCP autoimmunity in Greek patients with RA (smokers and non-smokers). Eighty-three (83) RA patients (41 smokers, 42 have never smoked) were typed for HLA-DRB1 by molecular techniques (PCR-SSOP and SSP). In 62 out of 83 (74.7%) anti-CCP abs were detected by ELISA. In RA patients in comparison to the controls, increased frequency of HLA-DRB1*01:01 (28.9% vs 6.8%, OR=4.4), *10:01 (16.9% vs 2.4%, OR=8.4), *04:01 (3.6% vs 2%, OR=1.8), *04:04 (7.2% vs 1%, OR=7.6) and *04:05 (15.7% vs 3.7%, OR=4.8), as well as decreased frequency of DRB1*04:02 (1.2% vs 2%, OR=0.6) and *04:03 (4.8% vs 6.8%, OR=0.7) was found. Among RA patients, 77.1 % possessed 1SE vs 18.9% of controls (OR=14.4), whereas 10.8 % possessed 2SE vs 1% of controls (OR=11.8). In CCP(+) RA patients in comparison to CCP(-), an increased frequency of HLA-DRB1*01:01 (27.4% vs 14.3%, OR=2.3) and *10:01 (21% vs 4.8%, OR=5.3) was observed. Furthermore, 88.7% of CCP(+) carried 1SE vs 42.9% of CCP(-) patients (OR=10.5). CCP(+) smokers patients and in comparison to CCP(+) non-smokers were presented with an increased frequency of DRB1*01:01 (41.9% vs 12.9%, OR=4.9). Among the CCP(+) smokers, 96.8% possess 1SE vs 80.6% of CCP(+) non-smokers (OR=7.2), whereas 12.9% possess 2SE vs 12.9% of CCP(+) non-smokers (OR=1). In conclusion: a) an increased frequency of HLA-DRB1*01:01, *10:01, *04:05 alleles, as well as the protective role of *04:02, *04:03 alleles, in Greek patients with RA was confirmed, b) the presence of any SE, particularly *10:01 allele, strongly influences the production of anti-CCP abs and c) interaction between smoking and any SE, particularly DRB1*01:01, is associated with anti-CCP positive RA in Greek patients.
HLA antigens and autoantibodies as screening test among family members of celiac disease patients
V. Kitsiou (Athens, GR)
Celiac Disease (CD) is a permanent intolerance to gluten, which in genetically susceptible individuals leads to bowel mucosal villous atrophy. It affects at least 1% of caucasoids, however is more common in certain high risk groups, such as family members (FMs) of CD patients. The aim of this study was the evaluation of serologic and genetic markers in investigation and assessment of familiar CD prevalence, in a Greek population. The study included 29 CD patients and 101 first-degree FMs not presenting with typical symptoms and signs of CD. HLA typing by high resolution PCR-SSP techniques for -DQA1/DQB1 alleles and serological identification of anti-tissue transglutaminase (tTG) by ELISA and anti-endomysial (EMA) autoantibodies (AAbs) by IIF, were performed. At least one CD predisposing HLA allele was typed in 28/29 (96.5%) patients and 75/101 (74.3%) FMs. More specifically 86 (66.1%) were DQ2 positive, 12 (9.2%) DQ8 positive and 7 (5.3%) DQ2/8 double positive. HLA-DQA1*05:01/DQB1*02:01 haplotype was present in 70 (53.8%), *02:01/02:02 in 19 (14.6%), *03:01/03:02 in 17 (13.1%), *03:03/02:02 in 7 (5.4%) and *03:02/03:02 and *03:01/03:05 in 1 (0.8%) individuals. All patients and 21 FMs were positive in at least one of the above AAbs and all of them but one patient carried a high risk HLA allele, 41 -DQ2 and 8 -DQ8. However, 56 individuals (43.1%) without positive AAbs expressed a high risk HLA allele. The study revealed 21 new CD cases (4 parents, 10 offspring, 7 siblings) according to ESPGHAN diagnostic criteria, all possessing both genetic and serological CD markers. The prevalence of CD among first-degree relatives (21/101) appears higher (20.8%) than in general population. Most of these patients had an atypical form of the disease and would therefore be overlooked, leading in serious complications. Therefore, a more pro-active screening strategy with HLA genotyping as well as tTG and EMA AAbs serological testing, should be strongly recommended in FMs of CD patients.
Lack of association of programmed death-1 gene polymorphisms rs2227981 and rs41386349 with multiple myeloma.
A. Witkowicz (Wrocław, PL)
Multiple myeloma (MM) is a B-cell malignancy characterized by the proliferation and an accumulation of isotype – switched immunoglobulin-producing monoclonal plasma cells in the bone marrow. A variety of T-cell abnormalities was reported in MM patients, such as marked reduction in the proportions of CD4 and CD8 cells expressing co-stimulatory molecules, signal transduction components, and Th1/Th2 imbalance. Therefore we focused our attention on one of the immune checkpoint molecules - Programmed Death 1 protein (PD-1). PD1 is a cell surface receptor belonging to the immunoglobulin super-family, which negatively regulates the immune response by limiting the activation and proliferation of T-cells. Expression of this transmembrane protein, its ligands and different genetic variations of PD-1 gene have been widely studied as a part of attempts to identify the pathogenesis of many diseases related to immune system. The aim of this study was to examine whether the PD-1 SNPs: rs2227981 C>T (PD-1.5) and rs41386349 C>T (p.7209), are genetic modifiers for risk of MM. Our case-control study was conducted in a Polish population consisting of 192 cases with multiple myeloma and 271 healthy individuals for PD-1.5, and 182 cases and 246 controls for p.7209. Genotyping was performed by polymerase chain reaction and restriction enzyme digestion: PvuII for PD-1.5 and MluI for p.7209. We found no significant differences in distribution of genotypes between MM patients and controls for both studied SNPs (rs2227981 C>T: CC - 32.3% vs 33.6%, CT – 51.6% vs 49.1%, TT 16.1% vs 14.4% and rs41386349 C>T: CC – 82.4 vs 86.6%, CT – 17.6% vs 13.0%, TT – 0% vs 0.4%). The haplotype analysis showed the trend toward higher risk of disease for patient with TT haplotype (OR=1.58; p=0.1). Our results indicates that rs2227981 C>T (PD-1.5) and rs41386349 C>T (p.7209) are not associated with the risk of multiple myeloma in Polish population, while the results should be confirmed on larger group of patients.
Association of polyglandular autoimmunity with HLA: it’s worthwhile to take a closer look into detail
B. Flesch (Bad Kreuznach, DE)
The association of glandular autoimmunity, e.g. type 1-diabetes (T1D) or autoimmune thyroid disease (AITD), with HLA class I and class II alleles is well documented. Data are more scarce concerning HLA class I alleles and polyglandular autoimmunity (PGA). HLA-B*08:01 has been associated with the development of PGA, but due to its high linkage to the susceptibility alleles DRB1*03:01, *04:01 and *04:04, the informative value is restricted. This study aimed to determine HLA class I allele association with PGA with a special impact on gender-related differences and discrimination for the involvement of AITD. HLA-A and HLA-B alleles of 143 PGA patients and 350 healthy controls were determined by PCR-SSO (Luminex, Immucor Lifecodes, Nijlen, Belgium) and PCR-SSP (Olerup, Vienna, Austria) on a 2-field level. Evaluation was performed separately for female and male patients and for the contribution of either Graves’ disease (GD) or Hashimoto’s Thyroiditis (HT). Although the HLA-B*40:01 allele frequency did not significantly vary between the healthy controls and the complete PGA patient collective it was associated with a significantly higher risk in female patients (RR=2.34; p= 0.018) while it proved to be protective for male patients (RR=0.20; p=0.005). This was also shown for PGA patients with a contribution of HT (PGA-HT) with a clearly enhanced risk for women (RR=3.20; p=0.008) and a protective effect for male patients (RR=0.15, p=0.024) while there was no difference for those patients with a contribution of GD. The B*50:01 allele was only over-represented in female PGA patients (RR=2.51; p=0.018) and especially in those women with PGA-HT (RR=3.45; p=0.005), while there was no significant difference in female PGA-GD patients. Gender related differences can result in a significantly different HLA class I allele association. This demonstrates the important influence of e.g. hormones and metabolic parameters on the development of polyglandular autoimmunity.
Stevens-Johnson Syndrome from allopurinol with HLA B*58
R. D'Auria (Avellino, IT)
To date there are studies on the association of HLA-B*58 with allopurinol-induced severe allergic dermatitis. It is known that this drug, mainly used for the treatment of hyperuricaemia, can cause the appearance of severe allergic reactions such as SJS and Lyell's syndrome in susceptible individuals. Typing of HLA-B can identify a predisposition to allergic reaction to allopurinol, avoiding the administration of this drug to patients with HLA-B*58. A 41 year old man in treatment for a month with allopurinol 300mg/day for hyperuricemia, came to our observation for the appearance of a rash on the palms of hands, soles of feet and subsequent commitment of the trunk and face. Erythematous lesions were presented as a rounded shade of deep red, with normal color in the center. These lesions were followed by desquamation of the lips and erythema of the oral mucosa making it difficult for the patient to feed himself. The patient was admitted to intensive care, where he was subjected to treatment with intravenous methylprednisolone 40mg and topical therapy with fluocortolone. On the 7th day the patient was discharged in good clinical condition. The HLA study of the HLA-B58*01 allele was positive. In another case, a 52 year old hypertensive man with familial hypercholesterolemia, who had been subjected to routine checks, was discovered to have high levels of uric acid. The hyperuricemia was subjected to therapy with allopurinol 300mg/day. On the 8th day he was hospitalized in E.R. for fever, erythema of the oral and conjunctival mucosa. Erythematous lesions on the skin, centered by a taut clear blister with negative Nikolsky’s sign gave the diagnosis of Stevens-Johnson Syndrome. The patient started intravenous and topical steroid therapy for the skin lesions. The HLA typing showed the presence of the HLA-B*58. HLA-B*58 is relatively rare in Caucasians. HLA typing is a useful tool for the protection of the patient's health and is a useful for the prevention of medical risk.
The KIR2DS1 receptor is a minor factor for susceptibility to psoriasis independent of HLA-C*06:02: a study of the Sardinian population
R. Littera (Cagliari, IT)
Psoriasis is a multi-factorial disease known to have a strong genetic susceptibility component. The HLA-C*06:02 allele has been suggested to be the most likely candidate for a predisposing role in disease pathogenesis since it has been found strongly associated with the condition in most populations worldwide. However, association of this allele is still a matter of controversy. Moreover, there are as yet no functional data supporting its pathogenetic role in psoriasis. In the study of psoriasis, killer-cell immunoglobulin-like receptors (KIRs), expressed by natural killer (NK) cells, have aroused considerable interest (KIRs) for their binding affinity with specific HLA-C molecules. Natural killer cells or natural killer-T (NKT) cells infiltrate psoriatic plaques and have been shown capable of causing the disease in immune-deficient murine models. In the present study, we compared the distribution of HLA-C and KIR genotypes (KIR2DS1, KIR2DS2, KIR2DL1, KIR2DL2 and KIR2DL3) between 340 Sardinian psoriatic patients and 200 healthy controls. Statistical analysis of the comparisons performed between the two groups revealed a significantly higher frequency of KIR2DS1 in the group of psoriatic patients (51% vs 41%; P=0.02). The KIRD2S1 locus was associated with psoriasis independent of the HLA-C*06:02 allele and did not seem to be influenced by clinical parameters such as: onset age, psoriasis area and severity index (PASI) or response to anti-tumor necrosis factor (TNF) drugs. These findings support the hypothesis that KIR2DS1 is a minor factor for susceptibility to psoriasis with a different mechanism of action compared to with respect to HLA-C*06:02.
Association of the MICA polymorphism with clinical response to anti-TNF therapy in rheumatoid arthritis patients
K. Bogunia-Kubik (Wroclaw, PL)
Major histocompatibility complex (MHC) class I chain–related A (MICA) proteins are stress-induced molecules involved in transmission of activating signals through NKG2D receptor. The MICA proteins are abberantly expressed on synovial tissue in rheumatoid arthritis patients.A total of 280 RA patients receiving anti-TNF therapy were enrolled to the study. Genotyping for MICA rs1051792 was performed using a polymerase chain reaction (PCR) amplification employing LightSNiP assays. Clinical response was evaluated according to the European League Against Rheumatism (EULAR) criteria at 12th and 24th week after initiation of the therapy. The MICA rs1051792 was significantly associated with clinical response after 12 weeks of anti-TNF treatment among patients. Patients carrying the heterozygous MICA rs1051792 AG genotype achieved significantly better EULAR responses to anti-TNF agents than patients with other genotypes (p=0.003). Moreover, inefficiency of anti-TNF therapy was more frequently observed after 12 weeks in patients with GG genotype than in patients bearing the A allele (p=0.010). No association was observed between MICA rs1051792 polymorphism and predisposition to RA development.These results imply that the MICA rs1051792 polymorphism may affect clinical response to therapy with TNF inhibitors in patients with RA.
A study on the correlation of HLA-E gene polymorphism with leukemia
Y. Xu (Shenzhen, CN)
We studied the correlation between the gene polymorphism of HLA-E and leukemia, with the aim of determining new molecular markers for the mechanism of leukemia occurrence and development. Peripheral blood DNA were extracted from patients with leukemia (n=59) and healthy individuals (n=132). The full-length HLA-E gene was amplified by long-range high-fidelity PCR and exon 3 was sequenced to identify the genotype of HLA-E. The genotype and allele frequency were calculated respectively in the leukemia patients and healthy controls. In the leukemia group, we detected 35 homozygous cases (59.3%), of which 29 (49.2%) were homozygous for HLA-E*01:03, and only 6 cases (10.2%) were homozygous for HLA-E*01:01. In healthy individuals, we detected 62 homozygous cases from healthy donors (47.0%), 36 cases (27.3%) were homozygous for HLA-E*01:03, 26 cases (19.7%) were homozygous for HLA-E*01:01. The allele frequencies of HLA-E*01:03 in leukemia patients and healthy individuals were 69.5% vs 53.7% (P=0.004, OR=2.0). The detection rate of HLA-E*01:03 homozygous in two groups were 49.2% vs 27.3 (P=0.003, OR=2.6). We concluce that HLA-E*01:03 is a susceptibility allele for leukemia.
Cytokine gene polymorphisms and clinical laboratory parameters in patients with multiple myeloma from the North-West Region of Russia
A. Pavlova (Saint-Petersburg, RU)
Polymorphic cytokine genes are involved in the pathogenesis of multiple myeloma (MM). We compared single nucleotide polymorphisms (SNP) of cytokine genes (IL-1a, IL-1b, IL-1R, IL-1RA, IL-2, IL-4, IL 4Ra, IL-6, IL-10, IL 12, TNF-a, IFN-g, TGF-b1) in 100 healthy unrelated Caucasoid blood donors with frequencies observed in 80 patients with MM (inhabitants of the same region). Genomic DNA was extracted from the peripheral blood; gene genotyping was performed by PCR-SSP, p-values less than 0.05 were considered statistically significant. The largest differences between two studied groups were found for IL-1 and IL-6. Frequencies of IL-1a -889 TT, IL-1b +3962 TT, IL-6 -174 GG, IL-6 nt565 GG in patients were higher than in donors (p<0.05). Further we analyzed albumin and beta2-microglobulin levels in the patients with/without homozygous genotypes for IL-1 and IL-6. All patients were divided into four groups: 1st- with IL-1a -889 TT, IL-1b +3962 TT, IL-6 -174 GG, IL-6 nt565 GG in their genotypes; 2nd – with IL-1a -889 TT, IL-1b +3962 TT; 3rd – with IL-6 -174 GG, IL-6 nt565 GG and 4th – without homozygotes in IL-1 and IL-6. Frequency of albumin low levels (up to 3.5 g/dl) was significantly higher in the 1-3 groups compare to the 4th group. The lowest levels of albumin (3.3 g/dl) was detected in the 1st group, however, the highest (more than 3.5 g/dl) was detected in patients without TT and GG homozygotes in IL-1 and IL-6 genotypes (p <0.05). The highest level of beta2-microglobulin was detected in the 1st group (5.5 mg/l) but no significant differences were detected compared to the other groups of patients (p> 0.05). Low levels of beta2-microglobulin weren’t registered in the 1st and 2nd group. The average level of beta2-microglobulin (3.5-5.5 mg/l) was frequently observed in the 4th group (0.70) and least registered in the 3rd group (0.42; p <0.05); high levels of protein (more than 5.5 mg/l) most rarely seen in patients from the 4th group (0.26).
Breast cancer: genetic risk or protection in Italian individuals.
A. Canossi (L’Aquila, IT)
Breast cancer (BC) is one of the most common and malignant diseases among women and hormonal and environmental factors could be involved in the presentation of BC. There is also evidence that genetic factors may favor or interfere with the occurrence of BC. The innate immune system with natural killer receptors (KIR) recognizing class I HLA molecules and tumor surveillance with HLA class II antigens presenting of tumor peptides to T lymphocytes are involved in anti-tumor immune response. The aim of this study was to investigate the association between the KIR genes and HLA-C alleles in Italian patients with BC and matched controls (Ctrs). In addition, we analysed the potential relationship between HLA-DRB1 alleles and cancer. Our SSP results regarding KIR and HLA-C gene polymorphisms (43 BC patients and 39 Italian female controls) showed a higher incidence of KIR2DS1 gene in advanced carcinoma patients (stage III-IV) compared to stage I-II patients (66.7% vs 28.0% p=0.0156; 46.1% in Ctrs). In particular, the KIR2DS1/HLA-C2+ combination was predisposing to a more advanced cancer (III-IV 42.9% vs I-II 6.3%, p=0.0309). On the contrary, KIR2DS4ins alleles seemed to be protective towards more advanced stages (11.1% vs 35.9% in Ctrs, p=0.06). In addition, HLA-DRB1 sequencing of 114 breast cancer patients and 120 female donors revealed the association of DRB1*11:03 allele with an increased risk to develop BC (p=0.025, OR=3.5674) and a protective role of HLA-DRB1*16:01 towards the BC cancer onset (p=0.019, OR=0.2328). Our findings suggest a potential role of KIR activating receptors licensed by HLA-C ligands in BC tumoral progression and an effect of some MHC class II alleles in the etiopathogenesis, suggesting that in the development of breast cancer exists a disorder of immune regulation.
Immunogenetic correlates of HIV in the North Indian population
N. Mehra (New Delhi, IN)
Genomic architecture of specialized cohorts of viremic controllers, elite controllers, exposed uninfected individuals, and rapid and slow progressor individuals indicate population specific genetic correlations of HIV/AIDS vulnerability. We performed candidate gene based studies to explore the genetic predisposition to HIV infection in a North Indian population. Particularly, we analyzed the genes that influence i) HIV cell entry (chemokine co-receptors like CCR5, CCR2 and their ligands like CCL3L1), ii) viral replication (tripartite interaction motif 5 a (TRIM5α), apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC), T cell/ transmembrane, immunoglobulin and mucin (TIM) family proteins, NFKB inhibitor like 1 (NFKBIL1) as well as iii) pro- and anti-inflammatory cytokines. Our major findings are (a) the well known protective variant CCR5 delta 32 is rare (<1%), and CCR5 promoter haplotypes were found associated with susceptibility and development of AIDS, (b) no association of CCR2 64I, CCL3L1 copy numbers and APOBEC3B deletion (29.5 kb) with HIV susceptibility and/or resistance, (c) among the observed seven TIM-1 exon-4 haplotypes, significantly higher CD4 counts were observed in D3-A +ve HIV patients, (d) TRIM5 α -exon 2 variant 43Tyr-allele and haplotypes carrying this allele were associated with resistance to HIV infection, e) low expressing allele *01 of NFKB IL1 gene and APOBEC3(H) haplotypes carrying rs139292 (N15del) and rs139297 (G105R) were associated with HIV susceptibility and (f) significantly higher allelic frequencies of IL-1α -889 T and IL-4 -1098 T were observed in HIV patients, while IL-1α -889 CC, IL-4 -1098 GG and IL-6 nt565 AA genotypes were observed significantly lower as compared healthy uninfected controls. The study represents distribution of immunogenetic variants and their influence on HIV/AIDS outcome in a North Indian population.
Inhibitory leukocyte immunoglobulin-like receptors LILRB1, 2, 3 and 4 on myelomonocytic cells and lymphocytes of patients with Behçet`s disease.
A. Kuranov (Göttingen, DE)
Behçet´s disease (BD), with a strong genetic risk association to HLA-B*51, represents a chronic multi-system vasculitis, in which increased chemotaxis and hypersensitivity of neutrophils could play an important pathogenetic role. The aim of this study was to investigate expression of inhibitory leucocyte Ig-like receptors LILRB1,2,3,4 which could down-modulate innate and adaptive immune responses by binding to classical and non-classical HLA-class I (LILRB1, B2), matrix proteins (LILRB3) and still unknown ligands (LILRB4) in BD patients and healthy controls. Expression of LILRB1-4 were investigated on myelomonocytic cells in comparison to lymphocytes in 21 BD patients and 21 healthy controls by indirect immunofluorescence labelling with specific monoclonal antibodies and FACS after specific density gradient separation. For data, statistical analysis using Flow Jo 7.2 software and graphical display of the results was performed using GraphPad Prism v5.0. Our results showed that LILRB1 expression was significantly reduced in patients (mean of LILRB1+ granulocytes: 17.3% in patients vs. 25.7% in controls p=0.0244) and mean LILRB1+ lymphocytes: 15.6% in patients vs. 27.4% in controls; p=0.0013). BD patients were also found to have significantly reduced expression of LILRB3 particularly on granulocytes (mean of LILRB3+ granulocytes: 12.1% in patients vs. 30.9% in controls; p<0.0001), but also on monocytes (mean of LILRB3+ monocytes: 13.3% in patients vs. 21.7% in controls; p=0.0134) LILRB4 was also shown to be significantly reduced on granulocytes of BD patients (mean of LILRB4+ granulocytes: 16.8% in patients vs. 26.2% in controls; p=0.0123). LILRB2 showed only a slight, but not significant weaker expression on myelomonocytic cells in BD patients. The analysis indicates that down-regulation of the expression of LILRB1, 3, 4 particularly on myelomonocytic could be involved in reduced inhibitory activity of specific innate immune cells in BD and thus participate in its pathogenesis.