Disease Association Studies Research Articles

Application of blood concentration biomarkers in nutritional epidemiology: example of carotenoid and tocopherol intake in relation to chronic disease risk

Biomarkers provide potential to objectively measure the intake of nutrients and foods, and thereby to strengthen nutritional epidemiology association studies. However, there are only a few established intake biomarkers, mostly based on recovery of nutrients or their metabolites in urine. Blood concentration measures provide a potential biomarker source for many additional nutritional variables, but their use in disease-association studies requires further development. The aim of this study was to apply recently proposed serum-based carotenoid and tocopherol intake biomarkers and to examine their association with the incidence of major cardiovascular diseases, cancers, and diabetes in a subset of Women's Health Initiative (WHI) cohorts. Serum concentrations of α- and β-carotene, lutein plus zeaxanthin (L+Z), and α-tocopherol were routinely measured at baseline in a subset of 5488 enrollees in WHI cohorts. Intake biomarkers for these 4 micronutrients, obtained by combining serum concentrations with participant characteristics, were recently proposed using a 153-woman feeding study within WHI. These biomarker equations are augmented here to include pertinent disease risk factors and are associated with subsequent chronic disease incidence in this WHI subset. HRs for a doubling of micronutrient intake differed only moderately from the null for the outcomes considered. However, somewhat lower risks of specific cardiovascular outcomes, breast cancer, and diabetes were associated with a higher intake of α- and β-carotene, lower risk of diabetes was associated with higher L+Z intake, and elevated risks of certain cardiovascular outcomes were associated with a higher intake of α-tocopherol. These patterns remained following the exclusion of baseline users of dietary supplements. Concentration biomarkers can be calculated from blood specimens obtained in large epidemiologic cohorts and applied directly in disease-association analyses, without relying on self-reported dietary data. Observed associations between carotenoid and tocopherol biomarkers and chronic disease risk could be usefully evaluated further using stored serum specimens on the entire WHI cohort. This study was registered at www.clinicaltrials.gov as NCT00000611.

Genetic signatures of gene flow and malaria-driven natural selection in sub-Saharan populations of the "endemic Burkitt Lymphoma belt".

Populations in sub-Saharan Africa have historically been exposed to intense selection from chronic infection with falciparum malaria. Interestingly, populations with the highest malaria intensity can be identified by the increased occurrence of endemic Burkitt Lymphoma (eBL), a pediatric cancer that affects populations with intense malaria exposure, in the so called “eBL belt” in sub-Saharan Africa. However, the effects of intense malaria exposure and sub-Saharan populations’ genetic histories remain poorly explored. To determine if historical migrations and intense malaria exposure have shaped the genetic composition of the eBL belt populations, we genotyped ~4.3 million SNPs in 1,708 individuals from Ghana and Northern Uganda, located on opposite sides of eBL belt and with ≥ 7 months/year of intense malaria exposure and published evidence of high incidence of BL. Among 35 Ghanaian tribes, we showed a predominantly West-Central African ancestry and genomic footprints of gene flow from Gambian and East African populations. In Uganda, the North West population showed a predominantly Nilotic ancestry, and the North Central population was a mixture of Nilotic and Southern Bantu ancestry, while the Southwest Ugandan population showed a predominant Southern Bantu ancestry. Our results support the hypothesis of diverse ancestral origins of the Ugandan, Kenyan and Tanzanian Great Lakes African populations, reflecting a confluence of Nilotic, Cushitic and Bantu migrations in the last 3000 years. Natural selection analyses suggest, for the first time, a strong positive selection signal in the ATP2B4 gene (rs10900588) in Northern Ugandan populations. These findings provide important baseline genomic data to facilitate disease association studies, including of eBL, in eBL belt populations.

Association of CD11b+ Monocytes and Anti–Vascular Endothelial Growth Factor Injections in Treatment of Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy

CD11b+ immune cells have been implicated in the formation of choroidal neovascularization in experimental studies on animals and disease-association studies on humans. However, the clinical importance of such observations remains unknown. To investigate whether the proportion of CD11b+ circulating monocytes is associated with the number of anti-vascular endothelial growth factor (anti-VEGF) injections in neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). These observational cohort studies collected data from January 1, 2010, through December 31, 2013, and from January 1, 2015, through December 31, 2018. Fresh venous blood samples were acquired for flow cytometric immune studies in patients with neovascular AMD or PCV receiving treatment with aflibercept or ranibizumab as needed for 36 months. Patients (n = 81) without immune diseases were consecutively recruited from a single center in Denmark. Proportion of CD11b+ circulating monocytes. The estimation of the number of intravitreal anti-VEGF injections given at 12, 24, and 36 months by the proportion of CD11b+ circulating monocytes and the correlation between these values. The angiogenic role of CD11b+ circulating monocytes was further evaluated by investigating the expression of the known proangiogenic receptor CCR2. Eighty-one patients were included in the analysis (54% women; mean [SD] age, 76 [7] years). The proportion of CD11b+ monocytes at baseline positively estimated the future number of anti-VEGF injections at 12 (ρ = 0.77; 95% CI, 0.35-0.93; P = .004), 24 (ρ = 0.82; 95% CI, 0.44-0.95; P = .002), and 36 (ρ = 0.78; 95% CI, 0.34-0.94; P = .005) months. This association was also found retrospectively in a larger sample of patients with neovascular AMD at 12 (ρ = 0.46; 95% CI, 0.16-0.68; P = .004), 24 (ρ = 0.49; 95% CI, 0.20-0.70; P = .002), and 36 (ρ = 0.65; 95% CI, 0.41-0.80; P < .001) months and patients with PCV at 12 (ρ = 0.27; 95% CI, -0.28 to 0.68; P = .30), 24 (ρ = 0.60; 95% CI, 0.12-0.85; P = .02), and 36 (ρ = 0.70; 95% CI, 0.27-0.90; P = .005) months, suggesting that this association is not specific to AMD but rather reflects VEGF activity in neovascularization. CD11b+ monocytes highly coexpressed CCR2, an important monocytic marker of proangiogenic activity. Results of this study demonstrated that the proportion of circulating CD11b+ monocytes estimated and correlated with the number of anti-VEGF injections in patients with neovascular AMD and PCV. Additional longitudinal studies are needed to determine whether these findings have clinical relevance to influence treatment algorithms or provide novel targets for medical therapy.

Ankylosing Spondylitis: A Trade Off of HLA-B27, ERAP, and Pathogen Interconnections? Focus on Sardinia.

The frequency of HLA-B27 in patients with Ankylosing Spondylitis (AS) is over 85%. There are more than 170 recognized HLA-B27 alleles but the majority of them is not sufficiently represented for genetic association studies. So far only two alleles, the HLA-B*2706 in Asia and the HLA-B*2709 in Sardinia, have not been found to be associated with AS. The highly homogenous genetic structure of the Sardinian population has favored the search of relevant variants for disease-association studies. Moreover, malaria, once endemic in the island, has been shown to have contributed to shape the native population genome affecting the relative allele frequency of relevant genes. In Sardinia, the prevalence of HLA-B*2709, which differs from the strongly AS-associated B*2705 prototype for one amino acid (His/Asp116) in the F pocket of the peptide binding groove, is around 20% of all HLA-B27 alleles. We have previously hypothesized that malaria could have contributed to the establishment of this allele in Sardinia. Based on our recent findings, in this perspective article we speculate that the Endoplasmic Reticulum Amino Peptidases, ERAP1 and 2, associated with AS and involved in antigen presentation, underwent co-selection by malaria. These genes, besides shaping the immunopeptidome of HLA-class I molecules, have other biological functions that could also be involved in the immunosurveillance against malaria.

Frequency of ABO/Rhesus (D) blood groupings and ethnic distribution in the Greater‐Accra region of Ghana, towards effective blood bank inventory

Knowledge on blood group phenotypes is of key importance in clinical practice. It used in blood transfusion practice to determine the direction of recruitment of voluntary donors as required for each population within a country, and for disease association and population genetics studies. This study aimed at reporting the frequency of ABO and Rhesus (Rh) groups in the population of the Greater-Accra region of Ghana and amongst their various ethnic groups. A retrospective study in 11 main hospitals within the region was done. Data collected provided information on the blood group status of persons (both blood donors and recipients) who visited the selected hospitals. Medical records used were within the years 2012-2017. A total of 42,317 (26,802 males and 15,515 females) data were retrieved and analysed. The frequencies of the blood groups O, A, B and AB were 50.0%, 24.3%, 20.7% and 5.0%, respectively. Rhesus-positive to negative ratio was 93.2%/6.2%. Frequencies of blood group O was highest (49.1%-53.6%) in all ethnic groups. The second most dominant blood group was B (24.2%-25.4%) in the Ga-Adamgbe, Akan and Ewe ethnic groups, whilst blood group A (25.0%-26.9%) was the second most dominant in the Northerners and non-Ghanaians. Blood group distribution amongst gender and different age groups showed no significant differences but followed the same pattern for the general population. The study provides data on the ethnic distribution and frequency of ABO and Rhesus blood groupings in the Greater-Accra region of Ghana. It also informs the need for blood banks in the region to increase the proportion of stockpiled Rhesus-positive blood groups especially for O, B and A that may be high in demand.

Overview of the BioCreative VI Precision Medicine Track: mining protein interactions and mutations for precision medicine.

The Precision Medicine Initiative is a multicenter effort aiming at formulating personalized treatments leveraging on individual patient data (clinical, genome sequence and functional genomic data) together with the information in large knowledge bases (KBs) that integrate genome annotation, disease association studies, electronic health records and other data types. The biomedical literature provides a rich foundation for populating these KBs, reporting genetic and molecular interactions that provide the scaffold for the cellular regulatory systems and detailing the influence of genetic variants in these interactions. The goal of BioCreative VI Precision Medicine Track was to extract this particular type of information and was organized in two tasks: (i) document triage task, focused on identifying scientific literature containing experimentally verified protein–protein interactions (PPIs) affected by genetic mutations and (ii) relation extraction task, focused on extracting the affected interactions (protein pairs). To assist system developers and task participants, a large-scale corpus of PubMed documents was manually annotated for this task. Ten teams worldwide contributed 22 distinct text-mining models for the document triage task, and six teams worldwide contributed 14 different text-mining systems for the relation extraction task. When comparing the text-mining system predictions with human annotations, for the triage task, the best F-score was 69.06%, the best precision was 62.89%, the best recall was 98.0% and the best average precision was 72.5%. For the relation extraction task, when taking homologous genes into account, the best F-score was 37.73%, the best precision was 46.5% and the best recall was 54.1%. Submitted systems explored a wide range of methods, from traditional rule-based, statistical and machine learning systems to state-of-the-art deep learning methods. Given the level of participation and the individual team results we find the precision medicine track to be successful in engaging the text-mining research community. In the meantime, the track produced a manually annotated corpus of 5509 PubMed documents developed by BioGRID curators and relevant for precision medicine. The data set is freely available to the community, and the specific interactions have been integrated into the BioGRID data set. In addition, this challenge provided the first results of automatically identifying PubMed articles that describe PPI affected by mutations, as well as extracting the affected relations from those articles. Still, much progress is needed for computer-assisted precision medicine text mining to become mainstream. Future work should focus on addressing the remaining technical challenges and incorporating the practical benefits of text-mining tools into real-world precision medicine information-related curation.

Protocols, Methods, and Tools for Genome-Wide Association Studies (GWAS) of Dental Traits.

Oral health and disease are known to be influenced by complex interactions between environmental (e.g., social and behavioral) factors and innate susceptibility. Although the exact contribution of genomics and other layers of "omics" to oral health is an area of active research, it is well established that the susceptibility to dental caries, periodontal disease, and other oral and craniofacial traits is substantially influenced by the human genome. A comprehensive understanding of these genomic factors is necessary for the realization of precision medicine in the oral health domain. To aid in this direction, the advent and increasing affordability of high-throughput genotyping has enabled the simultaneous interrogation of millions of genetic polymorphisms for association with oral and craniofacial traits. Specifically, genome-wide association studies (GWAS) of dental caries and periodontal disease have provided initial insights into novel loci and biological processes plausibly implicated in these two common, complex, biofilm-mediated diseases. This paper presents a summary of protocols, methods, tools, and pipelines for the conduct of GWAS of dental caries, periodontal disease, and related traits. The protocol begins with the consideration of different traits for both diseases and outlines procedures for genotyping, quality control, adjustment for population stratification, heritability and association analyses, annotation, reporting, and interpretation. Methods and tools available for GWAS are being constantly updated and improved; with this in mind, the presented approaches have been successfully applied in numerous GWAS and meta-analyses among tens of thousands of individuals, including dental traits such as dental caries and periodontal disease. As such, they can serve as a guide or template for future genomic investigations of these and other traits.

The complexity and diversity of major histocompatibility complex challenge disease association studies.

The major histocompatibility complex (MHC; 6p21.3) contains the most polymorphic genes, the most gene dense parts, and the highest diversity of functional gene clusters of the human genome. The clusters form haplotypes, which differ in linkage disequilibrium and show large variations in strength and extent between populations. Haplotype cis- and trans-expression quantitative trait loci have increased the knowledge of regulatory interactions between multiple MHC genes. The detailed haplotype data offer a reference for future studies in immune-mediated diseases and may unravel disease associations in conditions traditionally considered not to be immunologic. This article aims to describe the structural and functional variations of the MHC genes and haplotypes and their role on selected immune-mediated diseases. In immune-/inflammation-mediated complex diseases, hundreds of common variants influence the development of the disease trait, but the individual variants have small effects on the disease phenotypes as seen in genome-wide association studies. The genetic influence may still be significant on the cellular or molecular level. Nonetheless, the HLA alone is not sufficient as a susceptible genetic background to deduce the disease. For a comprehensive insight of the disease mechanisms, both immunological and genome assays methods are required.

Allele-Level KIR Genotyping of More Than a Million Samples: Workflow, Algorithm, and Observations.

The killer-cell immunoglobulin-like receptor (KIR) genes regulate natural killer cell activity, influencing predisposition to immune mediated disease, and affecting hematopoietic stem cell transplantation (HSCT) outcome. Owing to the complexity of the KIR locus, with extensive gene copy number variation (CNV) and allelic diversity, high-resolution characterization of KIR has so far been applied only to relatively small cohorts. Here, we present a comprehensive high-throughput KIR genotyping approach based on next generation sequencing. Through PCR amplification of specific exons, our approach delivers both copy numbers of the individual genes and allelic information for every KIR gene. Ten-fold replicate analysis of a set of 190 samples revealed a precision of 99.9%. Genotyping of an independent set of 360 samples resulted in an accuracy of more than 99% taking into account consistent copy number prediction. We applied the workflow to genotype 1.8 million stem cell donor registry samples. We report on the observed KIR allele diversity and relative abundance of alleles based on a subset of more than 300,000 samples. Furthermore, we identified more than 2,000 previously unreported KIR variants repeatedly in independent samples, underscoring the large diversity of the KIR region that awaits discovery. This cost-efficient high-resolution KIR genotyping approach is now applied to samples of volunteers registering as potential donors for HSCT. This will facilitate the utilization of KIR as additional selection criterion to improve unrelated donor stem cell transplantation outcome. In addition, the approach may serve studies requiring high-resolution KIR genotyping, like population genetics and disease association studies.

Genome-wide association meta-analysis yields 20 loci associated with gallstone disease

Gallstones are responsible for one of the most common diseases in the Western world and are commonly treated with cholecystectomy. We perform a meta-analysis of two genome-wide association studies of gallstone disease in Iceland and the UK, totaling 27,174 cases and 736,838 controls, uncovering 21 novel gallstone-associated variants at 20 loci. Two distinct low frequency missense variants in SLC10A2, encoding the apical sodium-dependent bile acid transporter (ASBT), associate with an increased risk of gallstone disease (Pro290Ser: OR = 1.36 [1.25–1.49], P = 2.1 × 10–12, MAF = 1%; Val98Ile: OR = 1.15 [1.10–1.20], P = 1.8 × 10–10, MAF = 4%). We demonstrate that lower bile acid transport by ASBT is accompanied by greater risk of gallstone disease and highlight the role of the intestinal compartment of the enterohepatic circulation of bile acids in gallstone disease susceptibility. Additionally, two low frequency missense variants in SERPINA1 and HNF4A and 17 common variants represent novel associations with gallstone disease.

Expanded Genome-Wide Association Study Meta-Analysis of Psoriasis Expands the Catalog of Common Psoriasis-Associated Variants.

Psoriasis is a complex immune-mediated disease of skin and joints with a prevalence of about 2%. Previous genetic association studies have identified more than 60 psoriasis susceptibility loci, 47 of which were identified in Caucasians. To further understand the genetic architecture of this disease, we conducted the largest meta-analysis of genome-wide association studies (GWAS) for psoriasis to date, including data from eight different Caucasian cohorts (seven GWAS and one Immunochip dataset), with a combined effective sample size of more than 39,000 individuals. Approximately half (n= 22) of the 47 European-origin loci were identified in cohorts containing a large proportion (≥50%) of samples genotyped using the Immunochip (Bowes etal., 2015; Stuart etal., 2015; Tsoi etal., 2012a, 2015), a platform that focuses on genetic variants from promising signals identified in previous association studies of autoimmune diseases (Stuart etal., 2015). However, restricting analysis to markers genotyped (∼110,000) (Tsoi etal., 2012b) or well-imputed (∼700,000) (Tsoi etal., 2015) on the Immunochip limits exploration of the full genome for susceptibility loci.

Genetic variants influence on the placenta regulatory landscape.

From genomic association studies, quantitative trait loci analysis, and epigenomic mapping, it is evident that significant efforts are necessary to define genetic-epigenetic interactions and understand their role in disease susceptibility and progression. For this reason, an analysis of the effects of genetic variation on gene expression and DNA methylation in human placentas at high resolution and whole-genome coverage will have multiple mechanistic and practical implications. By producing and analyzing DNA sequence variation (n = 303), DNA methylation (n = 303) and mRNA expression data (n = 80) from placentas from healthy women, we investigate the regulatory landscape of the human placenta and offer analytical approaches to integrate different types of genomic data and address some potential limitations of current platforms. We distinguish two profiles of interaction between expression and DNA methylation, revealing linear or bimodal effects, reflecting differences in genomic context, transcription factor recruitment, and possibly cell subpopulations. These findings help to clarify the interactions of genetic, epigenetic, and transcriptional regulatory mechanisms in normal human placentas. They also provide strong evidence for genotype-driven modifications of transcription and DNA methylation in normal placentas. In addition to these mechanistic implications, the data and analytical methods presented here will improve the interpretability of genome-wide and epigenome-wide association studies for human traits and diseases that involve placental functions.

Gender-specific associations between polymorphisms in the Toll-like receptor (TLR) genes and lung function among workers in swine operations

ABSTRACTWorkers in swine operations are exposed to dust, bacteria, and virus, and are at increased risk of respiratory problems. Toll-like receptors (TLR) play an important role in human immune responses to respiratory hazards. Worker gender and age may significantly modify the involvement of TLR in the etiology of these respiratory outcomes. The aim of this study was to investigate whether modification effects of worker gender and age altered associations between polymorphisms in the TLR genes and lung function. This study included 374 full-time workers from large swine operations from Saskatchewan. Information on demography, lifestyle, pulmonary function, and blood samples were obtained. Multiple linear regression and decision tree model were used in the analysis. Among females aged <45.8 years, workers with polymorphisms of rs4696480 in the TLR2 gene exhibited markedly better lung function than workers with wild-type. These associations were not observed among female workers aged ≥45.8 years and males. Among males, workers with polymorphisms of rs187084 in the TLR9 gene displayed significantly lower lung function than those with wild-type. This male-specific association was not dependent on worker age. This is the first study to report gender-specific correlations between lung function and polymorphisms of TLR genes, and modification effects of worker age on these associations, suggesting the importance of considering gender and age in genetic association studies of airway diseases due to exposure of high concentration of respiratory hazards.

Computational and Statistical Analysis of Array-Based DNA Methylation Data.

The characterization of aberrant DNA methylation is emerging as a key part of the study of cancer development and phenotype. The technical advancements and decreasing costs of methods for high-throughput profiling of DNA methylation have brought about a high interest in the use of such methods in disease association studies. Here we discuss the principles for DNA methylation analysis using data from the Infinium DNA methylation BeadChip assays and describe the computational steps and statistical considerations going from processing of the raw array data to analysis of differential methylation. Moreover, we provide detailed guidelines on how to perform tumor subtype classification based on DNA methylation signatures.

Identifying Key Genes of Liver Cancer by Networking of Multiple Data Sets.

Liver cancer is one of the deadliest cancers in the world. To find effective therapies for this cancer, it is indispensable to identify key genes, which may play critical roles in the incidence of the liver cancer. To identify key genes of the liver cancer with high accuracy, we integrated multiple microarray gene expression data sets to compute common differentially expressed genes, which will result more accurate than those from individual data set. To find the main functions or pathways that these genes are involved in, some enrichment analyses were performed including functional enrichment analysis, pathway enrichment analysis, and disease association study. Based on these genes, a protein-protein interaction network was constructed and analyzed to identify key genes of the liver cancer by combining the local and global influence of nodes in the network. The identified key genes, such as TOP2A, ESR1, and KMO, have been demonstrated to be key biomarkers of the liver cancer in many publications. All the results suggest that our method can effectively identify key genes of the liver cancer. Moreover, our method can be applied to other types of data sets to select key genes of other complex diseases.

Genetic Architecture of Adaptive Immune System Identifies Key Immune Regulators

SummaryThe immune system is highly diverse, but characterization of its genetic architecture has lagged behind the vast progress made by genome-wide association studies (GWASs) of emergent diseases. Our GWAS for 54 functionally relevant phenotypes of the adaptive immune system in 489 healthy individuals identifies eight genome-wide significant associations explaining 6%–20% of variance. Coding and splicing variants in PTPRC and COMMD10 are involved in memory T cell differentiation. Genetic variation controlling disease-relevant T helper cell subsets includes RICTOR and STON2 associated with Th2 and Th17, respectively, and the interferon-lambda locus controlling regulatory T cell proliferation. Early and memory B cell differentiation stages are associated with variation in LARP1B and SP4. Finally, the latrophilin family member ADGRL2 correlates with baseline pro-inflammatory interleukin-6 levels. Suggestive associations reveal mechanisms of autoimmune disease associations, in particular related to pro-inflammatory cytokine production. Pinpointing these key human immune regulators offers attractive therapeutic perspectives.

Seronegative Spondyloarthritis: Epidemiology, Pathogenesis, And Pathology

Classification of spondyloarthritis (SpA) is aimed at including patients with radiographic evidence of sacroiliitis and those with early disease who do not yet meet radiographic criteria but have positive features on magnetic resonance imaging (MRI). Most studies report a prevalence of SpA of 0.1 to 0.6%. Human leukocyte antigen (HLA)-B27 contributes approximately 20% of the heritability of SpA, and non–major histocompatibility complex loci identified to date (n = 113) contribute another approximately 10%. To date, 160 subtypes of HLA-B*27 have been reported, although population-based disease association studies are limited to only a few subtypes. Subtypes HLA-B*27:05 and HLA-B*27:04 are examples of subtypes associated with disease, whereas HLA-B*27:06 and HLA-B*27:09 are nonassociated. Properties of the B27 molecule relevant to pathogenesis include antigen presentation, propensity to misfold, and formation of homodimers. Key pathways identified by genetic studies include the interleukin (IL)-23 and M1-aminopeptidase pathways. The latter pathway is involved in peptide trimming in the endoplasmic reticulum, changing both the length and amino acid composition of peptides available for HLA class I presentation. IL-23 is a key cytokine regulating expression of IL-17 in a specific T helper cell phenotype, Th17, and also a variety of cells of the innate immune system. The IL-23–IL-17 pathway has been directly implicated in inflammation at sites that are inflamed in SpA. Increasing evidence based on prospective clinical and imaging data supports a link between inflammation and ankylosis, especially if the resolution of inflammation is followed by the appearance of a particular type of reparative tissue, namely, fat metaplasia, on T1-weighted MRI. This review contains 8 figures, 5 tables and 33 references Key words: association, classification, genetics, heritability, innate immunity, prevalence, spondyloarthritis

P075Extended HLA haplotypes in a greek population by NGS technology

Aim HLA alleles are observed in specific haplotypes due to Linkage Disequilibrium (LD) between certain alleles. Recent improvements in Next-Generation Sequencing (NGS) technologies accommodate the tight linkage disequilibrium between genes as well as the high degree of polymorphism of the MHC. In order to assign HLA haplotypes at allele level (4-fields) in a Greek population, in the context of 17th IHWS, 25 families including parents and siblings (95 subjects), all of Greek origin, were typed at 11 loci (HLA-A*, -B*, -C*, -DRB1*, DRB3/4/5*, -DQA1*, -DQB1*, -DPA1* and -DPB1*) by NGS technology. Methods Instrument and Chemistry: Illumina MiSeq, v2 chemistry, 300 cycles (2X150) and standard flow cell size. Illumina TruSight HLA v2.0 and Omixon Holotype HLA kits were used. HLA haplotype count and frequency (%) were estimated according to the number of parental haplotypes identified in the whole cohort. Additionally, haplotype count in families (N = 25) and subjects (N = 95) were calculated. Results HLA-A*01:01:01:01-C*07:01:01:01-B*08:01:01:01-DRB3*01:01:02:01-HLA-DRB1*03:01:01:01/02-DQA1*05:01:01:02-DQB1*02:01:01-DPA1*01:03:01:02-DPB1*04:01:01:01/02 haplotype was found in 3 members of 2 families (haplotype frequency 2%). All other haplotypes were observed once, giving haplotype frequency = 1%. Conclusions It is confirmed that the HLA-A*01-Cw*07-B*08-DRB1*03:01-DQB1*02 haplotype, defined by low-resolution molecular techniques, is the most common in Greeks, as in all Caucasians. These results are useful as reference data (controls) in disease association studies as well as in population genetics.

OR24 HLA allele and haplotype frequencies characterized using next-generation sequencing methods in unrelated world-wide populations: Summary from the 17th international HLA and immunogenetics workshop

Aim The primary goal of the 17th IHIWS was to advance the field of Histocompatibility and Immunogenetics research as well as clinical practices through the application of NGS technologies. Analyses of very-high resolution NGS HLA profiles sampled from different populations will be useful for reconstructing human history and may improve match predictions for unrelated donor selection algorithms. Methods In the unrelated population HLA diversity project, 15 laboratories from 10 different countries contributed HLA genotype data generated by various NGS methods. HLA population data included well-defined large datasets from the USA and East Asia and smaller samples from Europe, Australia, and Western Asia. Participants uploaded data in HML/XML format into the 17th IHIW database, data was systematically reviewed, and statistical analysis was performed using the genetic software PyPop v.0.7.0 to estimate; (i) allele frequencies, (ii) heterozygosity, (iii) conformity to HWE, (iv) selective neutrality, (v) 2-locus haplotypes and (vi) LD. The software BIGDAWG was used to estimate extended haplotype frequencies by an EM algorithm. Inter-population variance was determined using Nei’s genetic distances which were then used to construct phylogenetic dendrograms. Results Amongst each population, test of selective neutrality often revealed an excess of heterozygous compared with neutral expectations. A comparison of the HLA genetic diversity estimated at the 4 and 2 fields revealed that diversity at the majority of loci, particularly for populations of European descent, was lower at the 2-field resolution. African descent populations had a lower number of distinct HLA alleles at class I and II loci compared to other groups but exhibited higher levels of heterozygosity. Two-locus haplotype analyses revealed distinct HLA associations at the 4 − field. Analyses of extended haplotype frequencies, estimated both in the presence and absence of DP loci, showed unique haplotypes that were common to each ethnic group although some haplotypes were shared amongst different ethnic groups. Conclusions This workshop project provided a vast amount of new information about HLA genetic diversity at the intronic level and will be a useful resource for anthropological studies, disease association studies and transplantation.

Seronegative Spondyloarthritis: Epidemiology, Pathogenesis, And Pathology

Classification of spondyloarthritis (SpA) is aimed at including patients with radiographic evidence of sacroiliitis and those with early disease who do not yet meet radiographic criteria but have positive features on magnetic resonance imaging (MRI). Most studies report a prevalence of SpA of 0.1 to 0.6%. Human leukocyte antigen (HLA)-B27 contributes approximately 20% of the heritability of SpA, and non–major histocompatibility complex loci identified to date (n = 113) contribute another approximately 10%. To date, 160 subtypes of HLA-B*27 have been reported, although population-based disease association studies are limited to only a few subtypes. Subtypes HLA-B*27:05 and HLA-B*27:04 are examples of subtypes associated with disease, whereas HLA-B*27:06 and HLA-B*27:09 are nonassociated. Properties of the B27 molecule relevant to pathogenesis include antigen presentation, propensity to misfold, and formation of homodimers. Key pathways identified by genetic studies include the interleukin (IL)-23 and M1-aminopeptidase pathways. The latter pathway is involved in peptide trimming in the endoplasmic reticulum, changing both the length and amino acid composition of peptides available for HLA class I presentation. IL-23 is a key cytokine regulating expression of IL-17 in a specific T helper cell phenotype, Th17, and also a variety of cells of the innate immune system. The IL-23–IL-17 pathway has been directly implicated in inflammation at sites that are inflamed in SpA. Increasing evidence based on prospective clinical and imaging data supports a link between inflammation and ankylosis, especially if the resolution of inflammation is followed by the appearance of a particular type of reparative tissue, namely, fat metaplasia, on T1-weighted MRI. This review contains 8 figures, 5 tables and 33 references Key words: association, classification, genetics, heritability, innate immunity, prevalence, spondyloarthritis