CD59 polymorphisms mark differential expression levels and are associated with increased susceptibility to pemphigus foliaceus in females
D. Augusto (Curitiba, BR)
Pemphigus foliaceus (PF) is a bullous autoimmune disease characterized by acantholysis and autoantibodies against desmosomal antigens (particularly desmoglein 1), accompanied by complement system (CS) activation and painful epidermal blisters similar to burn injuries. CD59 is an important regulator of the complement cascade final step, in addition to mediating signal transductions and T lymphocytes activation. CD59 has different transcripts due alternative splicing, suggesting the presence of regulatory sites in their non-coding regions. However, there are no reports regarding the CD59 non-coding polymorphisms and their effect on autoimmune diseases. We haplotyped six CD59 non-coding polymorphisms with a possible effect in alternative splicing and gene expression: rs861256, rs831625, rs831629, rs704701, rs1047581 and rs704697, in 157 patients and 215 controls by PCR-SSP. Differential gene expression was evaluated in 82 subjects by qPCR. Genotype distribution was in Hardy-Weinberg equilibrium. The variant rs861256-G was associated with increased CD59 mRNA expression levels (P=0.01) and PF susceptibility in women (OR=4.11, P=0.0001). We observed that female patients were more prone to developing generalized lesions (OR=4.3, P=0.009) and to not experience disease remission (OR=3.7, P=0.045). Genetic associations were also observed for rs831625-G (OR=3.1, P=0.007) and rs704697-A (OR=3.4, P=0.006) in Euro-Brazilian women, and for rs704701-C (OR=2.33, P=0.037) in Afro-Brazilians. These alleles constitute GGCCAA haplotype, which also increases PF susceptibility (OR=4.9, P=0.045) and marks higher mRNA expression (P=0.0025). In conclusion, higher CD59 transcriptional levels seem to be related to PF susceptibility, probably due to transcriptional changes or to the CD59 role in T cell signal transduction and in cytokine release. These results should be replicated in independent cohorts and our hypothesis further investigated in functional studies.
Genetic characterization of immunoglobulin gamma heavy chain (IGHG) gene segments in Brazilian urban and Amerindian populations suggests evidence of natural selection
D. Augusto (Curitiba, BR)
The immunoglobulin gamma heavy chain (IGHG) gene segments encode the constant regions of the four immunoglobulins G (IgG) heavy chains, which are responsible for the effector functions of the mature molecules. Polymorphism of IGHG has been associated with susceptibility to cancer, autoimmunity and infection. However, presently knowledge of the diversity of IGHG is mostly restricted to that assessed by serological methods, because it has not been systematically sequenced in worldwide populations. Moreover, genetic polymorphism of immunoglobulins is not well covered in genome-wide studies and genomic databases. We characterized the genetic diversity of three IGHG gene segments (IGHG1, IGHG2 and IGHG3) by sequence-based typing in 348 individuals from several Brazilian populations: Japanese-descendant (n=51), Euro-descendant (n=41) and in five isolated Amerindian populations, Guarani Kaiowá (n=51), Guarani Ñandeva (n=51), Guarani Mbyá (n=47), Kaingang from Rio das Cobras (n=48) and Kaingang from Ivaí (n=49). A total of 25 new alleles have been found, of which 10 have been confirmed by cloning and sequencing. The most frequent alleles in Japanese-descendants were IGHG1*02 (61%), IGHG2*03 (53%) and IGHG3*14 (49%); in Euro-descendants, IGHG1*03 (72%), IGHG2*02 (47%) and IGHG3*11 (71%), and, in Amerindians, IGHG1*02 (45 to 77%), IGHG2*03 (74 to 96%) and IGHG3*14 (84 to 99%). Neutrality tests were performed for each gene segment and population. Tajima’s D values showed evidence of purifying selection (-1.9; p < 0.001), and Fay and Wu’s H statistic suggested that IGHG polymorphism is possibly driven by positive selection in nearby regions (-3.8; p < 0.01). Further studies are needed to verify which evolutionary pressures shape the genetic diversity of these gene segments. However, description of such unique isolated populations is already providing information for disease association studies and insights in immunoglobulin population genetics and evolution.
Significant associations between long non-coding RNA polymorphisms and pemphigus foliaceus susceptibility
D. Augusto (Curitiba, BR)
Pemphigus foliaceus (PF) is an epidermal bullous autoimmune disease, characterized by presence of antibodies against desmoglein 1, a molecule important for cell adhesion. Genetic and environmental factors contribute to this complex disease, which is endemic in certain regions of Brazil. Long non-coding RNAs (lncRNAs) are transcripts more than 200 nucleotides in length and lack coding potential. Most lncRNAs are involved in gene expression regulation by interacting with DNA, other RNAs and proteins. lncRNA dysregulation and polymorphism have emerged as important co-players in the onset or progression of complex diseases, being reported in associations with differential risk to cancer autoimmunity and infection. We aimed to investigate whether SNPs (single-nucleotide polymorphisms) located within lncRNAs genes could be associated with differential susceptibility to pemphigus foliaceus. For this purpose we intersected data from lncRNASNP database with genotype data (Illumina Beadchip Human Core Exome-24) from 235 patients and 6,681 controls. After standard quality control, we found 2,080 SNPs mapped to lncRNAs in our data. We performed an association study applying a logistic regression using principal components as covariants to correct for possible population structure bias. We found 3 SNPs associated with pemphigus foliaceus with P<0.001. The three associated SNPs are located in regions close to protein coding genes. The strongest association was found on chromosome 2, SNP rs1542604, mapped on the lncRNA AC133785.2. Suggestive associations (P<0.01) were seen for other 25 SNPs. We showed, for the first time, that SNPs located at lncRNAs could be associated with pemphigus susceptibility. Moreover, our results suggested that lncRNAs may be among the factors that influence pemphigus pathogenesis. We expect that lncRNAs quantification and analysis of the functional impact of their polymorphism will help to clarify the biological significance of these associations.
Functional analysis of allelic typed donor natural killer cell Receptors KIR2DL1 and KIR3DL1 for improved outcome in stem cell transplantation
Y. El-Nahry (Berlin, DE)
Genomic studies on KIR-HLA ligand interactions reveal activity differences based on allelic polymorphisms of NK KIR receptors, leading to differences in functional activity. However, these variations in affinity are not completely understood. In our study, allelic typing was performed by sequence analysis of KIR2DL1 and KIR3DL1 in a modified version after Hou et al. (2007) and Belle et al. (2008) including 21 healthy donors. NK cell activity was assessed by the CD107-degranulation assay against L721.221 transfected cell lines expressing HLA-C group 1, -C group 2, HLA-Bw4 ligands or HLA negative L721.221 cells. KIR exon sequences were analyzed by GeneSearch software using the IPD-KIR database to assign allelic typing. KIR3DL1 positive donors were grouped into ´low´ and ´high´ affinity allele groups. KIR2DL1 sequence analysis included a total of 57 polymorphisms. Position 245 in exon 7 was selected to separate donors into two groups based on an arginine/cytosine-exchange (R245/C245). Flow cytometry of KIR3DL1 low and high affinity confirmed the low/high expression pattern of alleles. NK cells missing their specific ligand on the target cells in the C245-group (N=5) showed general higher activity levels compared to donors of the R245-group (N=7) in CD107-assay. Nine analysed donors were heterozygous for both (R/C245). One new polymorphism was found showing the triplet GGC, coding for the amino acid glycine. To correlate our findings with clinical outcome data from the Children’s Transplantation Centre Frankfurt, 60 additional family stem cell donors were sequenced for the KIR2DL1 245-polymorphism and stratified for KIR2DL1 arginine (R) (245R) *001, *002, *00301-03, *005, *008, *009, *010 RR or R+ grafts with less mortality and better progress free survival (PFS). Determining allelic polymorphism in combination with functional analysis will lead to better medical prediction of the NK effect and could fill the gap between theory and praxis of NK cell alloreactivity.
KIR3DL1, but not its ligands, confers low risk for the development of Acute Lymphoblastic Leukemia (ALL) In Mexican Mestizo patients
B. González (México City, MX)
ALL accounts for >30% of all malignancies diagnosed before the age of 15 years. The disease is multifactorial, and an important role for inherited genetic variation has been suggested for its development. A spectrum of HLA associations were reported and polymorphisms at the killer cell immunoglobulin-like receptors (KIR) influence ALL development as well. KIR genes present on NK cells, regulate their functions through interactions with their HLA ligands, and are also documented to be involved in the destruction of leukemic cells. We addressed this issue by performing a case-control study in Mexican Mestizo patients with ALL, looking for the involvement of KIR3DL1 and its Bw4, A23, A24 & A32 ligands with the development of ALL. DNA was extracted from peripheral blood with the Maxwell16 instrument from 137 patients (90% were children) and 356 healthy controls. KIR3DL1 was detected using a PCR-SSP technique. HLA typing was done using a Luminex PCR-SSOP method. Frequencies of KIR3DL1 and KIR-HLA ligands were compared between patients and controls, using the Chi2 test. A low frequency of 3DL1 in patients vs. controls (80.3% vs. 88.5%; p=0.018; OR=0.530) was shown; however, no difference was observed for Bw4 or for A23, A24 & A32 (p=0.24) or for 3DL1 with any of its ligands (p=0.34). We previously reported protection associated with KIR2DS4 (p=0.002), KIR2DL2 (p=0.001), KIR2DS2 (p=0.0001) and KIR2DS3 (p=0.002) but not KIR3DL1, having typed then, only 66 patients. However, when increasing the number to 137, KIR3DL1 was clearly shown to be a protective gene. We thus conclude, that 3DL1 confers a low risk for ALL development in Mexicans. These data agree with those reported in Chinese populations, but differ from data found in Canadian whites and Indians and in admixed Hispanics from California (which include different races). Activating KIR2DS1-C2, 2DS2-C1 and 3DS1-Bw4 were risk genes in Indians only. Ethnicity differences clearly influence the genetic distribution of KIR genes and their ligands, therefore, probably their associations too. Undoubtedly, KIR3DL1 plays an important role in the expression and clearance of ALL in Mexicans. Moreover, these results are consistent across different ALL phenotypes, as shown in white Canadians. The importance of typing a greater sample size of patients is evident.
Expressed KIR gene frequencies in normal Polish population and leukemia patients
J. Nowak (Warsaw, PL)
Killer- cell immunoglobulin-like receptor (KIR) molecules play a key role in cancer immunosurveillance. We performed KIR genotyping in a normal Polish population (N=330) and in acute myeloid leukemia (AML, N= 191) and acute lymphoblastic leukemia patients (ALL, N=114). In the normal population some framework genes (KIR2DL4, 3DL2, 3DL3, 3DP1) were common and remaining genes occurred with certain gene frequency pattern (2DL1-0.809, 2DL2-0.308, 2DL3-0.670, 2DL5-0.292, 2DP1- 0.809, 2DS1-0.233, 2DS2-0.324, 2DS3-0.166, 2DS4-0.754, 2DS5-0.158, 3DL1-0.773, 3DS1- 0.218). It is considered that normal variants of some genes are functional and non-expressed variants with deletions are not (2DL4 norm-0.478 and del-0.517, 2DS4 norm-0.181 and del-0.584, 3DP1 norm-0.782 and variant-0.165, 2DL5A expressed-0.211 and 2DL5B null-0.240). The association study revealed a strong protective role of normally expressed KIR2DL4 in AML (OR=0.43, p=0.000045, 95%CI 0.29-0.64) and ALL (OR=0.46, p=0.0013, 95%CI 0.29-0.74) but not in it's deletion variant. In a ALL similar protective role has been revealed for expressed variants of KIR2DS4 (OR=0.41, p=0.0018, 95% CI 0.23-0.72). These associations withstood the Bonferroni corrections for 15 KIR gene comparisons. Of note, the only so far reported ligand of KIR2DL4 is the non-classical HLA class I molecule HLA-G, leading to the inhibition of the cytolytic NK cell function. The protective role of functional KIR2DL4 can be related to licensing capacity of inhibitory KIR upon engagement to HLA-G or another unknown ligand.
Killer-cell immunoglobulin-like receptor (KIR) polymorphism In retinoblastoma: a case-control study
R. Aggarwal (Chandigarh, IN)
Retinoblastoma is the most common cancer of the eye in children. A higher disease burden is recorded in large populations, such as Asia and Africa. An association between KIR and retinoblastoma has not been yet beenreported. The aim here was to investigate the frequency of KIR genes in patients with retinoblastoma and compare with controls. Children with retinoblastoma and healthy controls were enrolled. DNA was extracted from peripheral blood samples of cases and controls. KIR genotyping was performed by polymerase chain reaction using sequence-specific primer assay. Nineteen KIR genes were investigated. These included: inhibitory (2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DL5B), activating (2DS1, 2DS2, 2DS3, 2DS4*FUL, 2DS4*DEL, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1) and pseudogenes (2DP1, 3DP1*FUL, 3DP1*DEL). The association was determined by chi-square or Fisher’s exact test. The significance was adjusted with Bonferroni correction; a p value lower than 0.0026 (0.05/19) was considered significant. The study was approved by the institutional ethics committee. Thirty two patients with retinoblastoma and 60 controls were enrolled. The mean age of patients was 71.2 ± 19.5 months (range: 5-96), with a M:F ratio of 1:1.1. Seven (22%) patients had bilateral retinoblastoma. Of the 19 KIR loci analyzed, a significant difference in cases as compared to controls was observed at 3DL1, 3DL2, 3DL3 and 3DS1 loci. The inhibitory KIR genes that were significantly less well represented in patients with retinoblastoma included, 3DL1 (p 0.0004), 3DL2 (p 0.0002) and 3DL3 (p 0.0023). In addition, there was a significant down regulation of activating KIR gene 3DS1 (p 0.001) in cases. There was a significant reduction in frequency of 3DL2 (p 0.0006) and 3DS1 (p 0.0007) in patients with unilateral retinoblastoma. No association was observed with bilateral disease. A protective effect of 3DL1, 3DL2, 3DL3 and 3DS1 KIR alleles was observed in patients with retinoblastoma. The study provides novel insights concerning distribution of KIR genes in patients with retinoblastoma and has implications for the development of innovative immunotherapies for this childhood cancer.
Analysis of educated KIR 3DL1 in a patient with Multiple Myeloma
G. Sortino (Catania, IT)
Multiple Myeloma (MM) is a hematological malignancy characterized by accumulation in the bone marrow of plasma cells that secrete a monoclonal immunoglobulin (Ig). For over thirty years, the relationship between NK cells and MM has been described in clinical and scientific studies. The available data suggest that NK cells play a key role in the immune response. However, such role could be lost due to strategies of "immune escape" used by the myeloma cells. It is well known that NK cells are subject to an "educational" process that ensures the formation of a repertoire of functional but "self-tolerant" NK cells through the interaction of their cell surface receptors with the HLA molecules (ligands C1, C2, Bw4). These receptors provide the educational signal. The aim of the present study was to analyze the prognostic impact of KIR expression of NK cells in MM patients and in healthy controls, to assess the connection, if any, between KIR genes and MM. KIR genes and their HLA ligand were analyzed in 30 healthy subjects (controls) and 69 patients (MM) using the Genotyping SSP and SSO kit. Patients and controls were divided into two groups according to the presence of KIR genes, KIR Ligand, educated KIR genes and KIR haplotype AA/Bx. The gene frequencies were also compared using the Fisher's t-test with GraphPad Prism software version 6:00. KIR 3DL1-edu was found to be significantly more prevalent among controls as compared to MM patients (76.7% vs 43.59%, p<0.0023; CI=95%). For MM patients, the AA and Bx genotype frequencies were, respectively, 24.6% and 74.4% with an A:B ratio of 1:3.05. As for the healthy controls, the AA and Bx genotype frequencies were, respectively, 33% and 67% with an A:B ratio of 1:2. Our data suggest that the lower frequency of an educated receptor (3DL1edu) may be associated with a reduced cytotoxicity of NK cells against myeloma cells.