Resolving unknown nucleotides in the IPD-IMGT/HLA database by extended and full-length sequencing of HLA class I and II alleles.

In the past, identification of HLA alleles was limited to sequencing the region of the gene coding for the peptide binding groove, resulting in a lack of sequence information in the HLA database, challenging HLA allele assignment software programs. We investigated full-length sequences of 19 HLA class I and 7 HLA class II alleles, and we extended another 47 HLA class I alleles with sequences of 5' and 3' UTR regions that were all not yet available in the IPD-IMGT/HLA database. We resolved 8638 unknown nucleotides in the coding sequence of HLA class I and 2139 of HLA class II. Furthermore, with full-length sequencing of the 26 alleles, more than 90 kb of sequence information was added to the non-coding sequences, whereas extension of the 47 alleles resulted in the addition of 5.5 kb unknown nucleotides to the 5' UTR and > 31.7 kb to the 3' UTR region. With this information, some interesting features were observed, like possible recombination events and lineage evolutionary origins. The continuing increase in the availability of full-length sequences in the HLA database will enable the identification of the evolutionary origin and will help the community to improve the alignment and assignment accuracy of HLA alleles.

Dual Role of PTPN22 but Not NLRP3 Inflammasome Polymorphisms in Type 1 Diabetes and Celiac Disease in Children.

Genetic polymorphisms in genes coding for inflammasome components nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain-containing protein 3 (NLRP3) and caspase recruitment domain-containing protein 8 (CARD8) have been associated with autoinflammatory and autoimmune diseases. On the other hand several studies suggested that NLRP3 inflammasome contributes to maintenance of gastrointestinal immune homeostasis and that activation of NLRP3 is regulated by protein tyrosine phosphatase non-receptor 22 (PTPN22). PTPN22 polymorphism was implicated in the risk for various autoimmune diseases including type 1 diabetes (T1D) but not for celiac disease (CD). The aim of our study was to evaluate the role of inflammasome related polymorphisms in subjects with either T1D or CD as well as in subjects affected by both diseases. We examined PTPN22 rs2476601 (p.Arg620Trp), NLRP3 rs35829419 (p.Gln705Lys), and CARD8 rs2043211 (p.Cys10Ter) in 66 subjects with coexisting T1D and CD, 65 subjects with T1D who did not develop CD, 67 subjects diagnosed only with CD and 127 healthy unrelated Slovenian individuals. All results were adjusted for clinical characteristic and human leukocyte antigen (HLA) risk. PTPN22 rs2476601 allele was significantly more frequent among subjects with T1D (Padj = 0.001) and less frequent in subjects with CD (Padj = 0.039) when compared to controls. In patients with coexisting T1D and CD this variant was significantly less frequent compared to T1D group (Padj = 0.010). Protective effect on CD development in individuals with T1D was observed only within the low risk HLA group. On the other hand, we found no association of NLRP3 rs35829419 and CARD8 rs2043211 with the development of T1D, CD or both diseases together. In conclusion PTPN22 rs2476601polymorphism was significantly associated with the risk of developing T1D in Slovenian population, while no associations of proinflammatory NLRP3 and CARD8 polymorphisms with T1D and CD were observed. Interestingly, the same PTPN22 variant protected from CD. We hypothesize that this effect may be mediated through the NLRP3 inflammasome activation.

When type 1 diabetes meets celiac disease.

Type1 diabetes (T1D) and celiac disease (CD) may occur together. HLA-DQ8 and DQ2 are key genetic risk factors in both. Overall, the patients with co-existing T1D and CD (T1D+CD) were shown to have HLA profile more similar to patients with T1D than those with CD. Slovenian patients with both diseases had the frequency of DQB1*02:01 (86.5%) very similar to patients with CD (83.8%) in contrast to the significantly different frequency of the same allele in patients with T1D (52.24%). The DQ2 conveyed higher risk for developing both T1D and CD in the same individual than DQ8. Additionally, the A1-B8-DR3-DQ2-MICA*008 ancestral haplotype (8.1AH) was over-represented in Slovenian (T1D+CD) patients, where B*08 was the most significant independent risk factor. Moreover, C*07, that is also present in the 8.1AH, could have an impact on the innate immunity rout of this susceptibility through interaction with KIRs. In the genotype context, the CD risk in T1D patients was associated with DR3-DQ2/DR3-DQ2, whereas the risk for T1D in CD patients was associated with DR3-DQ2/DR4-DQ8. Less frequent screening for the second disease related antibodies could be considered in patients with the first disease and low risk genotype for obtaining the second-one. Individuals carrying high risk DR3-DQ2/DR3-DQ2 or DR3-DQ2/DR4DQ8 are more likely to develop both autoimmune diseases together than individuals carrying any other HLA genotypes.

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT).

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible. In our component "Extension of HLA sequences by full-length HLA allele-specific hemizygous Sanger sequencing" we have used full-length hemizygous Sanger Sequence Based Typing to achieve this goal. We selected samples of which full length sequences were not available in the IPD-IMGT/HLA database. In total we have generated the full-length sequences of 48 HLA-A, 45 -B and 31 -C alleles. For HLA-A extended alleles, 39/48 showed no intron differences compared to the first allele of the corresponding allele group, for HLA-B this was 26/45 and for HLA-C 20/31. Comparing the intron sequences to other alleles of the same allele group revealed that in 5/48 HLA-A, 16/45 HLA-B and 8/31 HLA-C alleles the intron sequence was identical to another allele of the same allele group. In the remaining 10 cases, the sequence either showed polymorphism at a conserved nucleotide or was the result of a gene conversion event. Elucidation of the full-length sequence gives insight in the polymorphic content of the alleles and facilitates the identification of its evolutionary origin.

High-risk genotypes HLA-DR3-DQ2/DR3-DQ2 and DR3-DQ2/DR4-DQ8 in co-occurrence of type 1 diabetes and celiac disease.

Shared susceptibility alleles in the HLA region contribute to the co-existence of type 1 diabetes (T1D) and celiac disease (CD). The aim of our study was to identify HLA genotype variations that influence co-occurrence of T1D and CD (T1D + CD) and the order of their onset. Totally 244 patients, 67 with T1D, 68 with CD and 69 with T1D + CD, (split into "T1D first" and "CD first"), were analyzed. Control group consisted of 130 healthy unrelated individuals. Two-tailed Fisher's exact test was used for statistical analysis. The genetic background of Slovenian CD patients resembled more northern than southern European populations with DR3-DQ2/DR3-DQ2 (odds ratio [OR] = 19.68) conferring the highest risk. The T1D + CD was associated with DR3-DQ2/DR3-DQ2 (OR = 45.53) and even more with DR3-DQ2/DR4-DQ8 (OR = 93.76). DR3-DQ2/DR7-DQ2 played a neutral role in susceptibility for T1D + CD. The order of the onset of T1D or CD in patients with co-occurring diseases was not influenced by HLA risk genotype profile. DR3-DQ2/DR3-DQ2 was associated with an increased risk for developing CD in patients with T1D, whereas patients with CD carrying DR3-DQ2/DR4-DQ8 were at higher risk for developing T1D. In addition to other genetic factors including HLA class I alleles present on DR3-DQ2 extended haplotype, the second extended haplotype may moderate the risk for T1D + CD conferred by DR3-DQ2. Our results suggested that individuals carrying high-risk genotypes DR3-DQ2/DR3-DQ2 or DR3-DQ2/DR4-DQ8 would more likely develop both T1D and CD than either disease alone.

Introduction to statistical analysis of population data in immunogenetics.

We review the most classical questions addressed by the analysis of population data in immunogenetics. Basic genetics' definitions are reminded. Questions related to the population data itself (structure, missing values nomenclature, sampling) are developed first, and secondly, the population genetics questions (relevance of genetic parameters (phenotype, genotype allele and haplotype frequencies; genetic distances; linkage disequilibrium measures), methods and practical computing) are illustrated by immunogenetics polymorphisms. This article gives the essential of population immunogenetics on examples and key references. We underline the importance of population dimension in statistical analysis: structure of linkage disequilibrium and genetic diversity between populations may affect the power of the study or the interpretation of correlations between markers, genes and diseases, making population genetics both theoretical and very practical.

The contribution of HLA-Class II antigens in humoral non-response and delayed response to HBsAg vaccination.

The variability in the immune response modulated by HLA alleles may be an important factor for the induction of the protective effect of HBsAg vaccines. We present here the analysis of HLA-DRB1, DQB1 and DQA1 alleles and their combinations in the group of 36 individuals with poor humoral immmune response to HBsAg vaccination. Comparison with the control group, consisted of 60 randomly choosen healthy subjects, revealed that the DRB1*1601, DQB1*0502, DQA1*0102 haplotype is overrepresented in the group of hyporesponders and may therefore be regarded as a factor influencing poor antibody responsiveness. We observed that after revaccination two of three individuals who failed to develop anti-HBs antibodies carry the same phenotype DRB1*0101,DRB1*0301;DQB1*0501, DQB1*0201;DQA1*0101,DQA1*0501, which supports the conjecture that immunogenicity of the HBsAg vaccine depends on specific combination of HLA DR and DQ molecules on antigen presenting cells.

HLA-associated hemorrhagic fever with renal syndrome disease progression in slovenian patients.

Major histocompatibility complex (MHC) class I and class II genes regulate the balance between appropriate aggressive responses and invading pathogens while minimizing the destruction of host tissue. Several studies have shown that in hemorrhagic fever with renal syndrome (HFRS) patients, the disease outcome is determined by a complex interaction between the virus and immunopathologic and human genetic factors. In Slovenia, the severity of the disease caused by Puumala virus (PUUV) is significantly lower than that of HFRS due to Dobrava virus (DOBV). We have determined 23 different HLA-B and 12 different HLA-DRB1 types in Slovenian HFRS patients. Comparison of HLA frequencies between healthy individuals and HFRS patients showed no strong association with the susceptibility for hantaviral infection. Significant associations were recognized when the patient group was separated according to the virus responsible for the infection. DOBV-infected patients have a significantly higher frequency of HLA-B*35 than PUUV-infected patients. For HLA class II genes, the biggest difference between the PUUV- and DOBV-infected groups of patients was in HLA-DRB1*13, where this phenotype was more frequent in PUUV-infected patients, especially in the severe form of the disease. HLA-B*07 could play a protective role in PUUV-caused HFRS in the Slovenian population. Our study shows diverse associations of HLA molecules with DOBV- and PUUV-induced HFRS, and therefore, we presume that different hantaviruses are presented differently through the same HLA molecules and that this might lead to either a more severe or a milder form of the disease. In line with this idea, we have noticed that HLA-B*35 might be a genetic risk factor for DOBV infection in the Slovenian population.

Basiliximab versus daclizumab combined with triple immunosuppression in deceased donor renal transplantation: a prospective, randomized study.

BACKGROUND: In this prospective, randomized, open-label, single-center study, we compared the efficacy and safety of two anti-interleukin-2 receptor monoclonal antibodies combined with triple immunosuppression. METHODS: The adult recipients of at least one human leukocyte antigen-mismatched deceased donor renal graft on cyclosporine microemulsion, mycophenolate mofetil, and methylprednisolone were randomized to induction with basiliximab or daclizumab, given in standard doses. An intent-to-treat analysis of 1-year data assessed the incidence of acute rejections, graft function, patient and graft survival, and safety of this therapy. RESULTS: Two hundred twelve patients were studied. At 12 months, 11 (10.3%) and 10 (9.5%) patients experienced biopsy-confirmed first acute rejection in basiliximab and daclizumab groups, respectively. Estimated glomerular filtration rate was 69+/-19 mL/min/1.73 m in the basiliximab and 66+/-21 mL/min/1.73 m in the daclizumab group. Patient survival was 97.2% with basiliximab and 97.1% with daclizumab, and graft survival was 94.4% vs. 90.5%, respectively. Hospital treatment was required for 50 and 59 infections in basiliximab and daclizumab groups, respectively. One renal cell carcinoma of native kidney and one basal cell carcinoma were detected in the basiliximab group, and one melanoma of skin in the daclizumab group. One hypersensitivity reaction was observed with daclizumab. No significant differences were found between the groups. CONCLUSION: Basiliximab or daclizumab combined with triple therapy was an efficient and a safe immunosuppression strategy, demonstrated with low incidence of acute rejections, excellent graft function, high survival rates, and acceptable adverse event profile in adult recipients within the 1st year after deceased donor renal transplantation.